Abstract: A multiplex reverse transcription polymerase chain reaction（multiplex RT-PCR）method was developed for the simultaneous detection of five potato viruses，Potato virus X，Potato virus M，Potato virus Y，Potato virus A and Potato virus S. Five compatible sets of primers specific for each virus were designed based on conserved sequences of coat protein（CP）gene for multiplex RT-PCR assay. The crucial factors of multiplex RT-PCR including annealing temperature，number of the PCR cycles，extension temperature and primers concentration were optimized for the highest sensitivity and specificity. Five specific fragments were simultaneously amplified in uniplex PCR reaction. Their molecular weights were determined to be 138，213，369，468 and 657 bp. The sequence analysis indicated that the five virusesshared at least 97% homology with other related viruses. The sensitivity was shown to be capable of positively identifying the five viruses with ≥ 10-3 mg of freesia tissue sample. This multiplex RT-PCR protocol can simultaneously detect five potato viruses from field samples and plantlets with accurately，rapidity，sensitivity.

Key words: potato, virus, detection method, multiplex RT-PCR