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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 ecosystems and are of great economic importance. Representatives of forest-forming boreal coniferous species are species of the genus <i>Pinus</i>, including Siberian pine (<i>Pinus sibirica</i> Du Tour) and Scots pine (<i>Pinus sylvestris</i> L.), which are valuable and widely used woody plant species. The purpose of this research was to conduct an extended study of genetic diversity, genetic structure, and differentiation of <i>P. sibirica</i> and <i>P. sylvestris</i> populations under the conditions of their habitat in the Middle and Northern Urals. We studied twelve populations of two <i>Pinus</i> species using the inter-simple sequence repeat (ISSR)-based DNA polymorphism detection PCR method. Populations are characterized by relatively high levels of genetic diversity (<i>P. sylvestris</i>: <i>H_e</i> = 0.163; <i>n_e</i> = 1.270; <i>I</i> = 0.249; <i>P. sibirica</i>: <i>H_e</i> = 0.148; <i>n_e</i> = 1.248; <i>I</i> = 0.225). Analysis of the intrapopulation genetic structure reveals that the studied populations are highly differentiated (<i>P. sylvestris</i>: G_ST = 0.362; <i>P. sibirica</i>: G_ST = 0.460). The interpopulation component comprised 36% and 46% of the total genetic diversity for <i>P. sylvestris</i> and <i>P. sibirica</i>, respectively. Using various algorithms to determine the spatial genetic structure, it was determined that <i>P. sylvestris</i> populations form two groups according to their location at a certain altitude above sea level. <i>P. sibirica</i> populations form two clusters, with an additional subdivision of the two 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.