Potato virus Y (PVY) is considered as one of the most harmful virus infections of this crop. Thus, it is a topical problem to breed potato varieties resistant against a wide range of PVY strains and to create initial breeding material that will have a combination of resistance genes from different species. The aim of the study was: (1) to genotype a collection of 376 breeding lines (BL), developed from complex interspecific hybrids, using DNA markers of PVY resistance genes, (2) to identify accessions with markers of resistance genes from different species for subsequent use in marker assisted selection (MAS), (3) to evaluate the suitability of DNA markers of PVY resistance genes for genotyping BL developed through interspecific hybridization. It was ascertained that the markers most widely represented in the collection were RYSC3 of the Ryadg gene (49.7%), Ry364 and RAPD38-530 of the Rychc gene (50.5% and 45.2%, respectively), and Yes3-3A of the Rysto gene (29.8%). The markers Ry186 of Rychc and GP122/EcoRV780 of Ryf-sto were found only in some accessions. The frequency of occurrence of BL that had markers of PVY resistance genes from two different species varied between 2.7% (Yes3-3a marker of Rysto and both two markers of Rychc) and 8.5-9.0% (RYSC3 marker of Ryadg and both two markers of Rychc, or only Ry364 marker of this gene). In total, the collection was found to contain 134 BL (47.6%) with markers of resistance genes from two different species. A combination of four markers for three genes of different origin (Ryadg, Rysto and Rychc) was found in 27 BL (7.2%). Extreme resistance to PVY of most BL (302 out of 357) was obviously determined by the presence in them of the currently used resistance genes detected by DNA markers applied in the study. Nevertheless, a significant part of accessions (55 of 61) that did not have any markers was resistant to PVY. At the same time, 13 BL (3.5%) with the markers were susceptible to the virus. Such a level of discrepancies is considered as acceptable for the initial MAS of breeding material. The obtained data on the presence of the markers of PVY resistance genes of different origin and their combination in BL ensures a more effective use of such BL in breeding in comparison with the BL resistant to the virus, though lacking corresponding markers.