The influence of co-infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVYO (pathotype and serotype O), PVYNW (pathotype N and serotype O), and PVYNTN (pathotype and serotype N). Different diagnostic methods, including DAS-ELISA, multiplex RT-PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co-infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate- and host-dependent in form. The highest rates of virus transmission by single aphids were recorded with PVYNTN isolates, and the lowest ones with PVYO isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within-plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.