In 2012 and 2013 the presence of Potato virus Y on tomato in greenhouses and fields was analyzed. A total number of 176 samples displaying different type of symptoms: deformation, discoloration, necrosis of leaves and growth retardation were collected. The presence of viral infection using enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) was confirmed in 147 samples. Biological and genetic diversity of the virus isolates was also analyzed. The analyses revealed that the particular strains cause different symptoms on the tested plants such as Solanum lycopersicum cv. Moneymaker, S. melongena and Datura inoxia. In contrast to previous years, in which only isolates belonging to PVYNWi-P strains were observed, four different strains were identified: PVYNWi-P, PVYNN242, PVYNTN and PVYO. The isolates belonging to PVYNWi-P strain remained dominant in the population.