Based on ecological information, the distribution range of Tatra vole Microtus tatricus from Central European Carpathian Mountains is distinctly fragmented even at the level of individual mountain ranges. To investigate genetic differentiation between populations, we used 17 microsatellite loci to assess population genetic parameters in 83 Tatra voles from eight localities in Western and one in High Tatra Mountains in Slovakia, including a non-continuous temporal sample spanning from 1978 to 2008. Bayesian analyses of individuals resulted in five clusters, showing congruence between relatedness of sampled individuals and geographical origin. Clustering was supported with F-statistics that showed moderate to pronounced genetic differentiation between clusters, but it was not consistent with isolation by distance analysis. Pairwise comparisons between samples from consecutive years (1997–2008) revealed two cases of genetic differentiation that indicate genetic drift between generations associated with immigration. Recorded fluctuations in effective population size (Ne) might be attributed to the effects of immigration as they coincide with increased genetic differentiation. Estimated values of Ne are below the population size thresholds recommended to minimize inbreeding depression and maintain sufficient evolutionary potential. We observed multiple paternity in the Tatra vole by verifying at least two fathers of one litter.