BACKGROUND: Due to broad geographical and ecological distribution of Scots pine we witness the shaping of a significant species heterogeneity. There is a demand in researching the features of the genetic structure and differentiation of Scots pine populations in different parts of the range. MATERIALS AND METHODS: 12 populations were scrutinized with the use of ISSR markers. The genetic structure was assessed by estimating basic indicators of genetic diversity (the number of alleles per locus, the number of effective alleles, and the expected heterozygosity) and by the analysis of molecular variance (AMOVA). Genetic differentiation was assessed by Neis GST statistic, Mantel test, Principal Coordinates Analysis (PCoA), and creating a tree diagram. RESULTS: Populations that grow on the right bank of the Volga in the northern and central parts of the Volga Uplands are characterized by a higher genetic diversity (Na = 1.841.89; Ne = 1.341.39; He = 0.2170.241) and a lower subdivision (GST = 0.092). Populations that grow on the left bank proved lower rates of genetic variability (Na = 1.681.81; Ne = 1.271.35; He = 0.1740.218) while the divergence was higher (GST = 0.179). Much of the genetic variability is within the populations (more than 80%). CONCLUSIONS: The study determined differences in the genetic structure and the degree of differentiation of Scots pine populations, that grow on different banks of the Volga in the Middle and Upper Volga Regions.