Potato virus Y, as one of the most important potato viruses. Due to occurring mutations and recombination within and between strains, PVY exists as a complex of at least nine strains. Therefore, the identification of PVY and its different strains is very important for effective control strategies of the virus. In this study, the simultaneous detection of different PVY strains in potato fields of Isfahan and Chaharmahalva Bakhtiari provinces was carried out by multiplex RT-PCR. One hundred and fifteen potato leaf samples showing symptoms including mosaic, mottle, vein necrosis, stunting and chlorotic spots were collected from Isfahan province and Chaharmahal va Bakhtiari province. The samples were classified into four groups based on symptoms: the first group with yellow spot, the second group with mild mosaic and yellowing, the third group with vein necrosis and mild mosaic and the fourth group with yellows symptoms. Total RNA was extracted from the samples and cDNA was synthesized. PCR was carried out using universal and specific primer pairs for each strain. PVY detection was first performed using the PVYCPcEcoRII / PVYCPcBamHI PVY-specific primer pair. To detect PVY strains, the infected samples were then analyzed using PVY strain-specific primer pairs by the multiplex RT-PCR method. The results showed that recombinant PVY N W (B) is the most abundant strain (84 samples) in Isfahan and Chaharmahalva Bakhtiari provinces. The infected samples amplified 853 bp and 441 bp bands. Three samples also amplified three fragments expected sizes of 853 bp, 633 bp and 441 bp, indicating that these samples were infected with recombinant PVY N W (A). Two samples amplified the expecting banding patterns of 853 bp, 441 bp and 278 bp, which showed the infection of samples with a recombinant PVY strain known as PVY261-4 strain. The results of multiplex RT-PCR showed the presence of recombinant. However, major strains were not detected in two provinces. There was also no correlation between symptom type in the field and the PVY strains. To determine the nucleotide similarity of the amplified bands of our samples with those of other PVY strains available at GenBank, three samples, including ab3, ch7 and sh2 were selected based on the band pattern and type of symptoms and sequenced. Multiple alignment was performed using DNAMAN software ver. 4.02. Multiple alignment of sh2 sample with 278 bp,441 bp and 853 bp band pattern showed that 278 bp band and 853 bp band had the highest similarity with PVY261-4 strain, which was similar to the results of multiplex RT-PCR test. In multiple alignment, ab3 and ch7 samples with 441 bp and 853 bp band patterns showed the highest nucleotide similarity with the PVY261-4 based on nucleotide sequences of these two bands which was different from the results of the multiplex RT-PCR test. These results indicate that the annealing site of the primers is a more important factor in the specificity than the sequence of the regions amplified by them. Detection of the recombinant strains and not detecting the major strains of potato virus Y in this study, as reported in other countries, indicates that the variability mechanisms are active in PVY. Furthermore, our results showed that the multiplex RT-PCR method could be used as a sensitive, accurate, cost-effective and reliable method in the timely and simultaneous detection of PVY strains. Key words: potato virus Y, multiplex RT-PCR, Recombinant strains