The potato Solanum tuberosum L. has the greatest diversity of cultivated and wild relatives, whose genetic nature has been studied insufficiently. The aim of the present study was to examine the resistance to PVY in S. chacoense Bitt. and S. pinnatisectum Dun. and to search for DNA markers linked to resistance genes. For the first time, representative populations of two diploid tuber-bearing Solanum species were evaluated by the resistance to PVY and the presence of DNA markers linked to the Ry (extreme resistance) or Ny (hypersensitivity) genes localized on the long arm of chromosome 9. Nine accessions of S. chacoense and six accessions of S. pinnatisectum represented by 168 and 170 genotypes, respectively, from the VIR collection were assessed by resistance to artificial infection with PVY. The differences in segregation of two wild potato species into phenotypic classes with response to PVY infection have been established. The diversity of visible reactions of S. chacoense plants after PVY infection differs from the uniform type of symptoms observed in S. pinnatisectum. DNA analysis was performed in 170 S. chacoense genotypes and 44 S. pinnatisectum genotypes using the Ry186, S1d11, and CT220 markers linked to genes determining viral resistance in potato varieties, breeding clones, or species of the genus Solanum L. Most genotypes of S. chacoense and S. pinnatisectum have amplified CT220 marker linked to the Nxphu gene and the Sw-5 gene in the IvP35 line of the species S. phureja Juz. et Buk. and tomato, respectively. The STS Ry186 marker linked to the Rychc gene in Japanese varieties created on the basis of S. chacoense was identified in a small number of S. chacoense genotypes; however, no association between the DNA marker and resistance phenotype was detected. The CAPS S1d11/AcsI marker, which distinguishes between the PVY resistant and susceptible genotypes of both wild potato species, was developed. The plants of two wild-growing tuber-bearing species of Solanum L. were first examined for the presence of the GBSS gene-specific PCR marker (the gene encoding granules-bound starch synthase). A significant part of the genotypes (15% in S. pinnatisectum and 25% in S. chacoense) did not reveal the genetic marker regulating amylose biosynthesis in the forming starch granules. No significant differences in the ability to form tubers depending on the genotypes with or without GBSS marker, indicating the possible presence of an alternative allelic variant of the GBSSI gene in S. chacoense and S. pinnatisectum species, were detected.