A survey of Potato virus Y (PVY) was conducted in cultivated fields in six Iranian provinces between January 2005 to July 2007. Two hundred samples from potato and tomato were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for potyviruses. Almost one fourth of the samples were found to be infected by PVY. Analysis of these PVY-positive samples using three monoclonal antibodies (MAbs) facilitating the simultaneous detection of three main strains namely the ordinary (PVY(O)), strain (PVY(N)) and C (PVY(C)) strains. However, the fourth strain (PVY(NTN)) and some others recombinant isolates were also identified by molecular methods. Host range and symptoms analysis using sap inoculation of four different strains of PVY onto a range of plants revealed that the four strains showed biological properties that seemed to be consistent with their molecular grouping. Fourteen isolates of PVY were chosen based on the host and geographical location, primer specificity and serology for further biological and molecular characterisation. The coat protein (CP) and P1 genes and 3'-non-translated region (3'NTR) from 14 representative isolates were sequenced and analysed with the sequences available in GenBank. Composite analysis of the P1, CP and 3'-UTR sequences with all full genome sequences of PVY revealed that there are three potential strains of PVY in Iran, PVY(O), PVY(N)-W and PVY(NTN). Isolate KER.SA(N) was the most divergent of all the 14 isolates reacted with PVY(N) specific MAbs but grouped with PVY(O) strains in maximum likelihood phylogentic analysis. The PVY(NTN) isolates from Iran more closely related to the European than North American PVY(NTN) isolates.