A phylogeographic study of the large Japanese wood mouse, Apodemus speciosus, on Sado Island, Japan, was performed based on sequences of the mitochondrial cytochrome b gene (1,140 bp). Our previous study covered the entire species range and suggested that the mice on Sado Island are monophyletic, exhibiting two well diverged lineages throughout the island. The present data also supported two lineages (the average number of nucleotide difference was 11.4), showing a weak phylogeographic structure. Given the high sequence divergence observed, we assumed historically subdivided populations within the island. Bayesian coalescent analysis supported a dual-population model rather than that of one large population. The times to most recent common ancestor of all sequences were 293,000 years ago [ka; 95% highest probability density (HPD) 85–634 ka] and 292 ka (HPD 102–605 ka) for the one- and dual-population models, respectively. These results suggest that the populations have undergone repeated separations and reconnections, rather than being subdivided completely through time. Our results are in accordance with other paleogeographic and phylogeographic evidence from the island. The present study highlighted a unique system of producing and maintaining genetic diversity and suggested prehistoric colonization of the A. speciosus population on Sado Island, thus supporting the ancient origin of the mammalian fauna of Sado Island.