The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVY^N) and potato tuber necrotic group (PVY^NTN) contain segments of the PVY^O and the PVY^N genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVY^NTN at sites HC/Pro-P3, 6K2–NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVY^N isolates (termed PVY^N:O). Protocols for specific differentiation of the recombinant PVY^NTN and PVY^N:O from the non-recombinant PVY^N are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641 bp from the recombinant PVY^NTN and PVY^N:O. The second and third primer pairs exclusively amplified fragments of 448 and 290 bp, respectively from the recombinant PVY^NTN. In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290 bp) were amplified exclusively from the recombinant PVY^NTN, while only one fragment (641 bp) was amplified from the PVY^N:O isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVY^O and North American (NA)-PVY^N/NTN. For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity.