The genetic structure of gray-sided voles was investigated at a spatial scale of 2 km using mtDNA sequences. The control region (674bp) of 162 voles was sequenced and 18 haplotypes were identified. Within 0.5-ha trapping plots (n = 8), the number of haplotypes and gene diversity was significantly greater in males than in females. The fixation index among plots for females (FGP = 0.241) was 3 times as large as that for males (0.075), implying male-biased dispersal. A simulation analysis showed that the observed genetic structure in males could be generated by modifying the observed haplotype distribution of females by adding the effects of local male dispersal. Half of the pairwise FGP (15/28) showed significant differentiation in females, whereas almost none (1/28) were significant in males. Isolation by distance was observed in females, whereas no clear spatial pattern was observed in males. Most pairwise FGP for females were not significant in the short- and intermediate-distance classes (≤1.0 km) as with those for males, whereas all showed significant differentiation in the long-distance class (>1.0 km) for females, but not for males. These findings indicate that the extent of subpopulations within which individuals interact differs between sexes.