After we had obtained DNA as well from the three other male mummies in the sample—Yuya, Amenhotep III, and the mysterious KV55—we set out to clarify the identity of Tutankhamun's father. On this critical issue the archaeological record was ambiguous. In several inscriptions from his reign, Tutankhamun refers to Amenhotep III as his father, but this cannot be taken as conclusive, since the term used could also be interpreted to mean "grandfather" or "ancestor." Also, according to the generally accepted chronology, Amenhotep III died about a decade before Tutankhamun was born.
Many scholars believe that his father was instead Akhenaten. Supporting this view is a broken limestone block found near Amarna that bears inscriptions calling both Tutankhaten and Ankhesenpaaten beloved children of the king. Since we know that Ankhesenpaaten was the daughter of Akhenaten, it follows that Tutankhaten (later Tutankhamun) was his son. Not all scholars find this evidence convincing, however, and some have argued that Tutankhamun's father was in fact the mysterious Smenkhkare. I always favored Akhenaten myself, but it was only a theory.
Once the mummies' DNA was isolated, it was a fairly simple matter to compare the Y chromosomes of Amenhotep III, KV55, and Tutankhamun and see that they were indeed related. (Related males share the same pattern of DNA in their Y chromosome, since this part of a male's genome is inherited directly from his father.) But to clarify their precise relationship required a more sophisticated kind of genetic fingerprinting. Along the chromosomes in our genomes there are specific known regions where the pattern of DNA letters—the A's, T's, G's, and C's that make up our genetic code—varies greatly between one person and another. These variations amount to different numbers of repeated sequences of the same few letters. Where one person might have a sequence of letters repeated ten times, for instance, another unrelated person might have the same sequence stuttered 15 times, a third person 20, and so on. A match between ten of these highly variable regions is enough for the FBI to conclude that the DNA left at a crime scene and that of a suspect might be one and the same.
Reuniting the members of a family separated 3,300 years ago requires a little less stringency than the standards needed to solve a crime. By comparing just eight of these variable regions, our team was able to establish with a probability of better than 99.99 percent that Amenhotep III was the father of the individual in KV55, who was in turn the father of Tutankhamun.