In order to investigate further the allele frequencies of the adenosine triphosphate-binding cassette sub-family C11 (ABCC11) gene, which determines earwax phenotypes, among ancient popu- lations in Hokkaido, Japan, the single-nucleotide polymorphism and the 27 bp deletion in the ABCC11 gene were analyzed in ancient DNA extracted from 38 Epi-Jomon, 24 Jomon, and one Satsumon spec- imens excavated from various archaeological sites in Hokkaido. Of the specimens analyzed, 10 Epi- Jomon and three Jomon specimens were successfully genotyped. One homozygote for the 27 bp dele- tion was found first in one Epi-Jomon specimen. Including previously reported data on the Okhotsk people and modern Ainu, exact tests of population differentiation showed that allele frequencies of the ABCC11 gene between the Jomon people and Ainu were statistically significantly different, whereas those between the Jomon and Epi-Jomon people and those between the Epi-Jomon people and Ainu were not significantly different. This result indicates that the Ainu, direct descendants of the Jomon people, were genetically affected by populations who possessed high frequencies of allele A (recessive dry allele) of the ABCC11 locus after the Jomon period.