We investigated the diversity and phylogeography of mitochondrial DNA (mtDNA) in Japanese macaques (Macaca fuscata), an endemic species in Japan that has the northernmost distribution of any non-human primate species. DNA samples from 135 localities representing the entire range of this species were compared. A total of 53 unique haplotypes were observed for the 412-bp partial mtDNA control region sequence, with length variation distinguishing the two subspecies. Clustering analyses suggested two putative major haplogroups, of which one was geographically distributed in eastern Japan and the other in western Japan. The populations in the east showed lower mtDNA diversity than those in the west. Phylogeographical relationships of haplotypes depicted with minimum spanning network suggested differences in population structure. Population expansion was significant for the eastern but not the western population, suggesting establishment of the ancestral population was relatively long ago in the west and recent in the east. Based on fossil evidence and past climate and vegetation changes, we inferred that the postulated population expansion may have taken place after the last glacial period (after 15,000 years ago). Mitochondrial DNA showed contrasting results in both variability and phylogenetic status of local populations to those of previous studies using protein variations, particularly for populations in the periphery of the range, with special inference on habitat change during the glacial period in response to cold adaptation.