A multiplex reverse transcription polymerase chain reaction (M-RT-PCR) was developed for the simultaneous detection of three major potato viruses, Potato virus X (PVX), Potato virus S (PVS) and Potato leaf roll virus (PLRV), in potato leaves and tubers (stored and dormant). Primers were designed from the coat protein gene of all three viruses and were able to detect virus in leaf RNA dilutions of 1 : 1024 to 1 : 4096 and from tuber RNA dilutions of 1 : 256 to 1 : 1024. The sensitivity of virus detection in leaves was higher than in tubers. All three viruses were successfully detected from tissues sampled from the tuber cortex, perimedulla and pith, with all PCR bands being strongest from cortex tissues. The reliability of the M-RT-PCR to detect PVX, PVS and PLRV in tubers was determined by testing 95 samples of field, greenhouse and in vitro potato plantlets from Wuhan, China and comparing the results with enzyme-linked immunosorbent assays (ELISA). M-RT-PCR detected all infections found by ELISA in leaf and stored tubers. In freshly harvested tubers, M-RT-PCR proved more sensitive than ELISA, as indicated by an additional 3.2% of infections detected with the M-RT-PCR method.