The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.