The least weasel Mustela nivalis and the ermine M. erminea of Japan are considered relicts of the last glacial period. To study phylogeographic variation in these mustelines, fragments of the mitochondrial DNA control region were sequenced. In both species, the control region included tandem repeats of 10base motifs at the 3′ portion specific to the genus Mustela. Phylogenetic trees of the 5′ portion (581–584 bases) of M. nivalis indicated that haplotypes in the Hokkaido population clearly diverged from those in the Honshu population. Geographic pattern of intraspecific variations illustrated by this result is concordant with that by the result of a previous cytogenetic study, which revealed the karyotypic differentiation between the two populations. These findings might suggest that the Hokkaido and Honshu populations of M. nivalis were geographically isolated in advance of the formation of the Tsugaru strait between Hokkaido and Honshu. Distribution pattern of the mitochondrial DNA haplotypes within Hokkaido suggests that M. nivalis experienced the repeated changes of environments during glacial and inter-glacial periods of the Quaternary, or that this species immigrated from Sakhalin/Siberia into Hokkaido so recently. On the other hand, M. erminea exhibited a smaller sequence divergence between the Hokkaido and Honshu populations, suggesting a shorter geographic isolation than in M. nivalis.