We examined the gene sequences of mitochondrial cytochrome b (cyt b) in two Japanese wood mouse species, Apodemus speciosus (n = 89) and A. argenteus (n = 46), which are distributed on the four main islands of Japan (Hokkaido, Honshu, Shikoku, and Kyushu) and on the small islands surrounding them. Apodemus speciosus, the larger of the two species, showed substantial genetic variation, with a maximum of 3% sequence divergence, and remarkable phylogenetic subdivision with two major clades. One clade represents haplotypes from a central region, including Honshu, Shikoku, Kyushu, and their adjacent islands; the other clade includes haplotypes from Hokkaido and the peripheral islands, forming four subclades: a) Hokkaido, b) Sado Island, c) Satsunan Islands, and d) the Izu Islands. Sequence divergence among the four subclades was 1.0 to 1.5%, implying that A. speciosus colonized these geographic regions 0.2 to 0.3 million years ago, assuming a substitution rate of 2.4% per million years. The population on the Izu Islands has preserved haplotypes that are distinct from those in any other region, providing good evidence for the natural colonization of the volcanic islands of the Izu Islands. The cyt b sequence variation had no relation to the karyotypic dimorphism for the eastern (2n = 48) and western (2n = 46) geographic groups, between which a strict border exists at central Honshu. On the other hand, Apodemus argenteus, the smaller of the two species, showed a similar level of sequence divergence (maximum of 3%) but no substantial geographic differentiation: populations in Hokkaido, Sado, and Yakushima shared similar haplotypes with each of the central populations, suggesting that genetic exchanges occurred among the localities in the last 0.15 million years. The apparent genetic structure of the mitochondrial DNA found in the A. speciosus population might be caused solely by long-term existence in insular regions, presumably due to ecological superiority relative to A. argenteus.