The harbor seal (Phoca vitulina) and spotted seal (Phoca largha) are the main seal species around Hokkaido, Japan. While some investigations have been conducted on the ecology and morphology of these two species, there is a lack of genetic information. We studied variation in mitochondrial cytochrome b sequences in the two species. Fifteen haplotypes were observed in 39 harbor seals from Erimo, Akkeshi, and Nosappu, and 23 were observed in 31 spotted seals from Erimo, Akkeshi, Nosappu, Rausu, Yagishiri Island, and Hamamasu. Phylogenetic trees showed two harbor seal lineages: Group I contained primarily haplotypes from Erimo, and Group II contained haplotypes from Akkeshi and Nosappu. Because the Erimo population had fewer haplotypes and less nucleotide diversity than the Akkeshi and Nosappu populations, we considered it to be Isolated from the others. In contrast, genetic variance within populations of spotted seals (97.3%) was far higher than that among populations (2.7%), determined by analysis of molecular variance. There were no significant difference among the spotted seal populations, indicating the absence of distinct lineages around Hokkaido. The differences in the genetic population structure between the two species could have been generated by their ecological differences. This study provides basic genetic information on these seal species and will contribute to the conservation and management of fisheries and seals throughout Hokkaido.