Roberts was born in 1928 in Waycross, Georgia, the only child of Pernell Elven Roberts, Sr. (1907–1980), a Dr Pepper salesman, and Minnie (Betty) Myrtle Morgan Roberts (1910–1988). During his high-school years, Pernell played the horn, acted in school and church plays, and sang in local USO shows. He attended, but did not graduate from, Georgia Tech. Enlisting in 1946, he served for two years in the United States Marine Corps. He played the tuba and horn in the Marine Corps Band, and he was also skilled at playing the sousaphone and percussion. He later attended, also without graduating, the University of Maryland, where he had his first exposure to acting in classical theatre. He appeared in four productions while a student, including Othello and Antigone, but left school to act in summer stock.

In 1949, he made his professional stage debut with Moss Hart and Kitty Carlisle in The Man Who Came to Dinner at the Olney Theatre in Olney, Maryland. Later, he spent eight weeks at the Bryn Mawr College Theatre in Philadelphia, portraying Dan in Emlyn Williams' Night Must Fall and Alfred Doolittle in Bernard Shaw's Pygmalion.

Roberts moved to Washington, D.C., in 1950 and supported himself with a variety of jobs while performing with the Arena Stage Theater for two years. He performed in numerous productions, including Steinbeck's Burning Bright, The Adding Machine, The Firebrand, The Deletable Judge, The Taming of the Shrew ("Petruchio"), Playboy of the Western World, Children of Darkness, School for Wives, The Inspector General, The Glass Menagerie, Mr. Arcularis, Twelfth Night, The Scarecrow, The Importance of Being Earnest, Julius Caesar, She Stoops to Conquer, School for Scandal, Three Men on a Horse, Faith of Our Fathers (Sesquicentennial Amphitheatre) and Dark of the Moon.

Roberts married four times, first in 1951 to Vera Mowry — a professor of theatre history at Washington State University and subsequently Hunter College, as well as professor emerita of the Ph.D. program in theatre at City University of New York — with whom he had his only child (Jonathan Christopher "Chris" Roberts, b. October 1951). Pernell and his first wife later divorced. Chris Roberts, who lived variously in California and New York, attended Franconia College.

In 1952, Roberts moved to New York, where he appeared first off-Broadway in one-act operas and ballets with the North American Lyric Theatre, with the Shakespearewrights, at the Equity Library Theatre, and later on Broadway with performances in Tonight in Samarkand (also in Washington, DC), The Lovers opposite Joanne Woodward, and A Clearing in the Woods with Robert Culp and Kim Stanley. He won a Drama Desk Award in 1955 for his performance in an off-Broadway rendition of Macbeth, which was followed by the role of Mercutio in Romeo and Juliet. He performed in Twelfth Night, Merchant of Venice, Dr. Faustus, and The Taming of the Shrew at the American Shakespeare Festival, and later on Broadway. He performed in St. Joan (1954, Cleveland), Down in the Valley (at the Provincetown Playhouse), The Duchess of Malfi, Measure for Measure, and King John. .

In 1956, Roberts returned to the Olney Theatre, starring opposite Jan Farrand in Much Ado About Nothing with the Players, Inc. group. The same year, Roberts made his television debut in the "Shadow of Suspicion" episode of Kraft Television Theater, followed by guest-starring roles in Whirlybirds, Gunsmoke, Cimarron City, Buckskin, Sugarfoot, and Cheyenne.

Roberts signed a contract with Columbia Pictures in 1957 and made his film debut a year later as one of Burl Ives' contentious sons in Desire Under the Elms (1958). The film was nominated for a Best Cinematography Academy Award. He also landed a character role in The Sheepman (1958), opposite Glenn Ford and Shirley MacLaine, and continued to guest-star on television shows such as episodes of Shirley Temple Storybook Theater ("The Emperor's New Clothes", "Rumplestiltskin", "The Sleeping Beauty", and "Hiawatha"), the live-broadcast Matinee Theater, where he starred again in Shakespeare's Much Ado About Nothing, and in The Heart's Desire. This was followed by appearances in Trackdown, Buckskin, and episodes of Zane Grey Theater. Roberts guest-starred as Captain Jacques Chavez on the NBC adventure series Northwest Passage (1958), based on the life of Major Robert Rogers in the French and Indian War. He appeared with fellow guest star Fay Spain in the 1958 episode "Pick up the Gun" of Tombstone Territory and played the lead villain in the 31st episode ("Hey Boy's Revenge") of Have Gun – Will Travel, portraying a killer boss exploiter of Chinese coolie laborers.

In 1959, Roberts guest-starred in episodes of General Electric Theater, Cimarron City, Sugarfoot, Lawman, One Step Beyond, Bronco, 77 Sunset Strip, The Detectives, and House Call. Also in 1959, he co-starred in the film Ride Lonesome. "If Roberts felt typecast by Westerns, they also provided his finest role in this film, arguably the greatest of the B-films, starring Randolph Scott and directed by Budd Boetticher. Roberts recognized the film's classic structure; his engaging outlaw, Sam Boone, counterpoints Scott's granite-faced Ben Brigade, maintaining the tension of whether they will work together or clash. He similarly played off James Coburn, who was making his film debut as Boone's quiet sidekick, Whit." The same year, he was cast in Bonanza.

Roberts married Judith Anna LeBrecque on October 15, 1962; they divorced in 1971. He subsequently married Kara Knack in 1972, divorcing in 1996.

He did The Night of the Iguana while still playing in Bonanza in 1963.

Roberts, having largely been "a stage actor, accustomed as he was to a rigorous diet of the classics" and to freely move about from part to part, found the "transition to a television series", playing the same character, "without costume changes," a difficult one. "It was perhaps not surprising that, despite enormous success, he left Bonanza after the 1964–65 season, criticizing the show's simple-minded content and lack of minority actors...". It particularly distressed him that his character, a man in his 30s, had to defer continually to the wishes of his widowed father, and he reportedly disliked the series itself, calling it "junk" television and accusing NBC of "perpetuating banality and contributing to the dehumanization of the industry." The equally self-critical Roberts ("I guess I'll never be satisfied with my own work"), "had long disdained the medium's commercialization of his craft and its mass production, assembly-line mindset." Frustrated with Bonanza and angry, he told a reporter in 1965, "I feel I'm an aristocrat in my field of endeavor. My being part of Bonanza was like Isaac Stern sitting in with Lawrence Welk".

He narrated documentaries, including the National Geographic episode, "Alaska, The Great Land" in 1965, "In the Realm of the Alligator" in 1986, the TV special Code One about the work of paramedics in 1989, and "The Mountain Men" episode of the History Channel, 1999.

Roberts acknowledged reasons for Bonanza's appeal, but pointed to his personal need for story lines with greater social relevance, adult themes and dialogue. He wanted Bonanza to be "a little more grown up," (Mike Douglas Show, 1966). He also noted that he was not suited to the "procedural" and "confining aspect" of series television, another reason for his dissatisfaction, while on the show (Mike Douglas Show, 1966).

Roberts was the only accomplished singer of the original cast, though David Canary, who joined Bonanza in 1967, had a background in voice and performed on Broadway. During Roberts' Bonanza years, he recorded Come All Ye Fair and Tender Ladies, a folk music album which AllMusic calls "...the softer, lyrical side of folk music — pleasant and not challenging, but quite rewarding in its unassuming way." The album, released by RCA Victor and arranged by Dick Rosmini, is available on compact disc only as part of the fourth disc of the Bonanza 4-CD boxed set on Bear Family Records.

Roberts guest-starred in TV shows such as The Girl from U.N.C.L.E., The Virginian, The Big Valley, Lancer, Mission: Impossible, Have Gun Will Travel, Marcus Welby, M.D., The Wild Wild West, Ironside, The Rockford Files, Gunsmoke, Mannix, Vega$, The Odd Couple, Hawaii Five-O, The Love Boat, Hotel, The Hardy Boys/Nancy Drew Mysteries, San Francisco International Airport, Nakia, Night Gallery, The Bold Ones, The Quest, Police Story, Most Wanted, Westside Medical, Man From Atlantis, Jigsaw John, Sixth Sense, Quincy, M.E. The Feather and Father Gang, Hawkins, Men from Shiloh, Perry Mason, Wide World of Mystery, and The Six Million Dollar Man, and appeared in miniseries, including Captains and the Kings, Centennial, The Immigrants and Around the World in 80 Days. He starred in two cult films, Four Rode Out (1971) and Kashmiri Run (1970), directed by the veteran TV Director John Peyser, and other feature films, including The Magic of Lassie (1978). He co-starred or was featured in several TV movies, including, The Adventures of Nick Carter, Dead Man on the Run, Assignment: Munich, The Night Rider, The Silent Gun, The Lives of Jenny Dolan, The Deadly Tower, Hot Rod, Desperado, The Bravos, and High Noon, Part II: The Return of Will Kane.

In 1972, Roberts returned to Broadway and toured with Ingrid Bergman in Captain Brassbound's Conversion, in which he played the title role. "Particularly helpful is Pernell Roberts in the acted upon title role... This actor is a sturdy, not unamusing leading-man type and may his appearance as a Bergman costar be rewarded beyond Bonanza."

In 1973, Roberts was nominated for a Joseph Jefferson Award for his performance in Welcome Home at the Ivanhoe Theatre in Chicago.

In 1979, Roberts again achieved "superstar" status as the lead in Trapper John, M.D. (1979–1986), receiving an Emmy nomination in 1981; and playing the character twice as long as Wayne Rogers had (1972–1975) on CBS's M*A*S*H series. Roberts told TV Guide (1979) that he chose to return to weekly television after watching his father age and realizing that it was a vulnerable time to be without financial security. "The show allowed Roberts to both use his dramatic range and address issues," wrote The Independent.

In the 1980s and 1990s, playing off his Trapper John M.D. persona, Roberts acted as TV spokesman for Ecotrin, a brand of analgesic tablets.

In 1988, Roberts co-starred with Milla Jovovich in the TV movie The Night Train to Kathmandu.

Roles since included Donor (1990) with Melissa Gilbert and Checkered Flag (1990).

From 1991 to 1993, in his last venture into series television, Roberts lent his distinctive voice to host and narrate the TV anthology series, FBI: The Untold Stories.

He made his last TV appearance in 1997 on an episode of Diagnosis: Murder, updating a Mannix character he had portrayed decades before.

In 1967, Roberts starred in the lavish, but short-lived David Merrick production of Mata Hari, directed by Vincente Minnelli. The show had a much-publicized "chaotic" preview performance due to technical problems stemming from lack of rehearsal time at the Kennedy Center, Washington, DC, where the preview performance took place. "What was offered the people of Washington was a dress rehearsal. David Merrick spoke to the audience beforehand warning them of this." Problems were corrected by the official opening night, when the show received good reviews for Roberts, musical score and lyrics, stage design and costumes, but poor reviews for its co-star and other aspects of the production. The show, nevertheless, was thought to have the potential to continue to Broadway. "Mata Hari was a show with a great story, two fascinating characters, and some accessory mess that could have easily been tidied up by anyone but Vincente Minnelli." But Merrick, "instead of bringing someone to clean house closed the production down" ("Opening A New Window: The Broadway Musical in the 1960s," p. 205 Ethan Morrden, October 29, 2002).

At the time of his death from pancreatic cancer on January 24, 2010, Roberts was married to Eleanor Criswell.