The aim of this study is to determine the genetic variability and differentiation among populations of pedun-culate oak ( Quercus robur ) at the eastern margin of the species distribution range. We applied new set of 95 geo-graphically informative nuclear SNP (single nucleotide poly-morphism) loci developed using Double Digest Restriction Site Associated DNA (ddRAD), a new generation DNA sequencing technology. The study area is located in the basins of the Volga River and its two main tributaries, Kama and Belaya. Despite the strong reduction and fragmentation of pedunculate oak forests over the last several centuries and comparatively small sizes of present stands, a relatively similar genetic diversity of seven populations was observed (on average, allelic diversity υ а = 1.472–1.603, observed heterozygosity H O = 0.305–0.358, expected heterozygosity H E = 0.272–0.343). The genetic distances between popula-tions vary between 0.102 and 0.170 (on average, d 0 = 0.128). We have identified the absence of a spatial genetic struc-ture and the existence of genetically peculiar populations in the territory studied. The genetic distances of Gregorius between populations vary between 0.102 and 0.170 (on average, d 0 = 0.128). Genetic differentiation among popula-tions was statistically significant with a mean of the fixa-tion index F ST of 0.075. Populations from the Privolzhye Upplands and from northern parts of the study area make the most contribution to overall inter-population differen-tiation (d 0 = 0.137). No significant differences exist among the samples at lowlands along the Volga river (d 0 = 0.110). It was concluded that the multiplicity and different ages of local populations and the complexity of migration routes of the pedunculate oak from Pleistocene refugia and secondary Holocene shelters can cause this phenomenon.