Abstract The East European vole Microtus mystacinus is the most widespread vole species in Anatolia. It is also frequently seen in watery habitats in a large area of Eurasia. In this paper, an attempt was made to ascertain the level of genetic differentiation between Anatolian (Asian part of Turkey) and Turkish Thracian (European part of Turkey) populations together with additional data from other parts of Asia and Europe by analysing two mitochondrial (cytochrome-b and cytochrome oxidase subunit I) and one nuclear (interphotoreceptor retinoid binding protein) gene regions. Acquired Bayesian Inference trees mostly separated the Asian and European populations of M. mystacinus and the fixation index values implied a significant differentiation between these populations for mitochondrial DNA. On the other hand, the median-joining networks did not show diverging populations, significantly, and the mean genetic distance values among populations were found to be low for both mitochondrial and nuclear DNA. Evolutionary divergence times of Asian and European populations were also calculated and dated back to approximately 0.316–0.111 million years ago, coinciding with the ice ages of the Pleistocene epoch. According to the obtained results, M. mystacinus populations have not diverged enough to form different species; however, there is a separation between Asian and European populations which might result in speciation.