Geographic isolation is among the most important factors initiating speciation in mammals. The narrow-headed vole Lasiopodomys gregalis is a widely distributed species in steppe habitats of South Siberia that are highly fragmented by mountain ranges. There are three major mostly allopatric mitochondrial lineages of L. gregalis described throughout the distribution range that most plausibly split in the Middle Pleistocene. This study applies the analysis of five microsatellite loci and species distribution modelling to understand the patterns of isolation and migration between L. gregalis populations in South Siberia and in particular in Tuva basin where all three lineages occur simultaneously. Microsatellite analysis supported the population differentiation revealed previously, with three clusters corresponding to mitochondrial lineages and indicated that gene flow between them is strongly limited. There were only traces of hybridisation in the population near the contact zone of lineages B and C. SDM analysis predicted only a small number of potential contact zones between L. gregalis lineages and barriers corresponding to landscape heterogeneity. We presume that the forested mountain ranges act as the main insurmountable barriers to gene flow between major genetic lineages of L. gregalis.