Nevertheless, the KV21a mummy has DNA consistent with the 18th dynasty royal line, therefore making it likely she was a member of the Thutmoside ruling house and supporting her identification with Ankhesenamun.

The identity of the queen who wrote the letter is uncertain. She is called Dakhamunzu in the Hittite annuals, a possible transliteration of the Egyptian title Tahemetnesu (The King's Wife). Possible candidates are Nefertiti, Meritaten, and Ankhesenamun. Ankhesenamun seemed once likely since there were no candidates for the throne on the death of her husband, Tutankhamun, whereas Akhenaten had at least two legitimate successors. but this was based on a 27-year reign for the last 18th pharaoh Horemheb who is now accepted to have had a shorter reign of only 14 years. This makes the deceased Egyptian king appear to be Akhenaten instead rather than Tutankhamun. The phrase regarding marriage to 'one of my subjects' (translated by some as 'servants') is possibly a reference to the Grand Vizier Ay or a secondary member of the Egyptian royal family line. Since Nefertiti was depicted as powerful as her husband in official monuments smiting Egypt's enemies, she might be the Dakhamunzu in the Amarna correspondence as Nicholas Reeves believes. Ankhesenamun may have been pressured by Ay to marry him and legitimise his claim to the throne of Egypt (which she eventually did) This also might explain why she describes herself as 'afraid', especially considering the popular (but not widely accepted) theory that Ay had a hand in her husband's death. A CT scan taken in 2005 shows that he had badly broken his leg shortly before his death, and that the leg had become infected. DNA analysis conducted in 2010 showed the presence of malaria in his system. It is believed that these two conditions, malaria and leiomyomata, combined, led to his death. However, Ankhesenamun was not as powerful as Nefertiti to be able to choose her spouse from a foreign state.

DNA testing announced in February 2010 has speculated that her mummy is one of two 18th Dynasty queens recovered from KV21 in the Valley of the Kings.

She is believed to have been born in Waset (present-day Thebes), but probably grew up in her father's new capital city of Akhetaten (present-day Amarna). The three eldest daughters – Meritaten, Meketaten, and Ankhesenpaaten – became the "Senior Princesses" and participated in many functions of the government and religion. Her birthdate is not known.