Sex-biased dispersal (SBD) is usually accounted for by the consequences of resource competition, inbreeding avoidance, and breeding competition. Although SBD has been reported in many mammalian species, little is known about it in subterranean species, like moles, whose dispersal from a natal place cannot be easily studied using conventional methods such as direct observations and radio-telemetry. Here, we used a genetic approach to study the population structure of the lesser Japanese mole Mogera imaizumii and tested whether SBD occurs in these solitary moles. We collected samples from three local populations in northern Japan and developed 12 novel microsatellite markers for genetic analyses. Population-based analyses showed a significant genetic differentiation among the populations. In individual-based analyses, we tested the degree of SBD using two different approaches: genetic relatedness between individuals and corrected assignment index (AIc). There was no significant difference in mean genetic relatedness and mean AIc between male and female groups in all three populations, although the proportion of individuals with positive AIc was higher in females than males in all three populations. The new microsatellite markers will help understanding of population genetic structure and behavior of moles in the further study.