The plant immunity is aimed at protecting against biotic and abiotic stresses and, therefore, at adapting to adverse environmental conditions. At the first protection step, a wide range of phytopathogenic receptors encoded by resistance R-genes is employed. The presence of a conserved NBS-domain in the receptors makes it possible to profile the plant genome by amplification of R-gene analogues. The method of multilocus NBS-profiling makes it possible to efficiently characterize the plant genome in terms of the representativeness and variability of the NBS-domain containing R-genes. This method is used to study the diversity of R-gene loci in crops and related wild species, as well as the introgressive hybridization phenomena and the R-gene evolution in plant species with varying degrees of pathogen resistance. NBS-profiling is also applied for genotyping GenBank collections, developing markers and saturating genetic maps. The requirement for cultivar genotype certification and profiling, along with a limited number of similar studies in Russia, makes research on the molecular profiling of domestic and foreign cultivars farmed in the Russian Federation relevant. In the present work, NBS-profiling was used for genotyping 65 potato Solanum tuberosum cultivars of mainly modern domestic breeding, as well as the related species Solanum stoloniferum (as an outgroup). Using two primer/enzyme combinations (NBS7/MseI and NBS9/MseI), 204 NBS fragments were generated, of which 144 (70.6 %) were polymorphic and one fragment was unique to cv. Gala. For each cultivar, a specific spectrum of NBS fragments was determined. Analysis of genetic distance matrix revealed a high level of polymorphism (GD = 0.18-0.45 with an average value of 0.33) among the studied cultivars. Genetic distances within the analyzed cultivars varied more than between the cultivars and the accession of S. stoloniferum (GD = 0.27-0.40). The most related cultivars were Solnechny/Pamyati Rogacheva (GD = 0.18) and Velikan/Vympel (GD = 0.19) originated from Lorch Potato Research Institute, and the most distant cultivars were Charoito/Red Scarlett (GD = 0.45). Statistical analysis of NBS-profiling data clustered studied potato cultivars in accordance with the pedigree and resistance to phytopathogens. On the dendrogram and graphs generated using the PAST and Structure 2.3.4, a pronounced tendency to group cultivars by traits of resistance to the Potato virus Y (Potyvirus, Potyviridae) and the Potato leafroll virus (Polerovirus, Luteoviridae) was shown. The primer/enzyme systems used in this study for NBS-profiling can be applied to study the mechanisms of potato resistance to biotic stresses.