The sustainable use and conservation of forest resources must be carried out with a detailed study of the main forest-forming plant species. Coniferous forests form the basis of boreal forest eco-systems and are of great economic importance. One of the representatives of forest-forming boreal coniferous species is species of the genus Pinus, including Siberian pine (Pinus sibirica Du Tour) and Scots pine (Pinus sylvestris L.), which are valuable and widely used woody plant species. The purpose of the study was an extended study of genetic diversity, genetic structure, and differen-tiation, of P. sibirica and P. sylvestris populations under the conditions of their habitat in the Middle and Northern Urals. We studied twelve populations of two Pinus species using the Inter Simple Sequence Repeats (ISSR) based DNA polymorphism detection PCR method. Populations are characterized by relatively high levels of genetic diversity (P. sylvestris: He = 0.163; ne = 1.270; I = 0.249; P. sibirica: He = 0.148; ne = 1.248; I = 0.225). Analysis of the intra-population genetic structure reveals that the studied populations are highly differentiated (P. sylvestris: GST = 0.362; P. sibirica: GST = 0.460). The interpopulation component comprised 36% and 46% of the total genetic diversity, for P. sylvestris and P. sibirica, respectively. Using various algorithms to determine the spatial genetic structure, it has been determined that P. sylvestris populations form 2 groups, according to their location at a certain altitude above sea level. P. sibirica populations form 2 clusters, with additional subdivision of the 2 populations into subclusters identified. The data obtained during the study may be useful for further research as well as for conservation management planning and related forestry practices aimed at preserving the genetic resources of valuable forest plant species.