To study the effects of post-glacial isolation by islands on population genetic diversity and differentiation of the large Japanese field mouse, Apodemus speciosus, we examined partial nucleotide sequences of the mitochondrial Dloop region (ca. 300 bp) in 231 individuals collected from islands in the Seto Inland Sea and adjacent regions on Honshu and Shikoku Islands in the western part of the Japanese archipelago. Molecular phylogenetic and network analyses showed that haplotypes in each island tended to form monophyletic groups, while those in Honshu and Shikoku (the major Japanese islands) showed scattered relationships and were connected with island haplotypes. These observations suggest that a set of Honshu and Shikoku haplotypes became the ancestral lineages of the island population. No gene flow was detected among island populations, indicating that independent evolution occurred on each island, without the influence of human activities, since the establishment of the islands in the Holocene. Population genetic diversities on each island were lower than those on Honshu and Shikoku. Comparison between genetic diversity and island area size showed positive correlations and supported the suggestion that genetic drift is a major factor that shaped the current haplotype constitution of the islands in the Seto Inland Sea.