During the Pleistocene in the northern part of Europe and Asia, the presence of ice sheets not only limited the range of species but also influenced landscape and thus the contemporary habitat system that determines the pattern of biodiversity. The aim of the research was to find out whether and how a lowland landscape, which formed as a result of subsequent Pleistocene glaciations (five) that in Eurasia covered various and generally successively smaller areas, affected the genetic differentiation of a species. The research was carried out in eastern Poland on the root vole Microtus oeconomus (Arvicolinae, Rodentia), a model boreal and hygrophilous species. Samples were collected from 549 vole individuals at 33 locations. Based on the analysis of 12 microsatellite loci and the 908 bp of cytochrome b sequences (mitochondrial DNA), the genetic structure of M. oeconomus in the landscape zones of the Polish Lowlands was determined. The results show that the latitudinal variability of the relief in eastern Poland (resulting from different ranges of Pleistocene ice sheets) and the related specific configuration of hydrogenic habitats are reflected in the genetic differentiation of the root vole. Therefore, it may be concluded that the history of landscape development affects the genetic structure of hydrophilic species.