In this study, we successfully extracted ancient DNA from skeletal remains of the Japanese sea lion—a species that is practically extinct—from archaeological sites and determined a partial sequence of its mitochondrial DNA control region. A molecular phylogenetic tree constructed by the neighbor-joining (NJ) method showed that the sequences from Japanese sea lions clustered together, with a high bootstrap value, and that this cluster was closest to the California sea lion cluster. The distinctly divergent cluster of Japanese sea lions reflected the morphological classification of these animals as a distinct species of the genus Zalophus; however, proximity to the California sea lion cluster simultaneously implied conformation with the traditional classification of these animals as a subspecies of Zalophus californianus. The average amount of nucleotide substitution between the Japanese and California sea lions was 7.02%. The Japanese and California sea lions were estimated to have diverged 2.2 million years ago, i.e., in the late Pliocene Epoch. This is the first report on a genetic analysis of the Japanese sea lion.