Abstract: A single-step multiplex reverse transcription-polymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection and discrimination among five RNA viruses namely Apple cholorotic leaf spot virus (ACLSV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Plum pox virus (PPV) and Tomato ringspot virus (ToRSV) which considered the most economically damaging viruses of stone fruit trees in Egypt and worldwide. Five compatible primer sets for one-step RT-PCR amplification were used in a single closed tube to detect the presence of virus infection. Five fragments with different sizes corresponding to the different viral targets were amplified from infected samples, i.e., 677, 172, 300, 220 and 490 bp for ACLSV, PDV, PNRSV, PPV and ToRSV, respectively. Results showed that multiplex PCR products of the expected sizes were obtained for all virus isolates assayed when they were tested either alone or in combination in an artificial mixture with no interference. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using this multiplex method, many samples can be simultaneously analyzed and it can be used routinely for sanitary and certification programmes.