We performed a phylogeographic analysis of the Japanese hare, Lepus brachyurus, using the mitochondrial cytochrome b gene (1140 bp). In total, 119 haplotypes were recovered from 197 samples isolated from 82 localities on three main islands of the Japanese archipelago: Honshu, Sikoku, Kyushu, Sado Island and the Oki Islands. Results showed two distinct clades at a genetic distance of 3.5%, equivalent to an estimated 1.2 million years. The two clades, encompassing seven subclades, showed an apparent geographic affinity to Kyushu, Shikoku and the nearby area of Honshu (southern group) by one clade, whereas the other clade covered the remaining area of Honshu (northern group). The landscape shape interpolation analysis exhibited a higher genetic diversity in the southern parts of central Honshu (northern group) and Shikoku and Kyushu regions (southern group), suggesting the existence of multiple geographical origins of population expansion in each clade. The Bayesian skyline plot analysis showed that lineage diversifications occurred about 0.35, 0.20 and 0.05 million years ago (Mya), which coincide closely with the glacial—interglacial cycles during the Pleistocene. Therefore, we suggest that the Japanese hare population once inhabited northern and southern refugia, and subsequently developed several populations through local demographic fluctuations. The present day demarcation in the northern and southern geographic groups is considered to be a temporal remnant of Pleistocene population dynamics and the geographic boundary between them could move or fade away in time.