tA multiplex RT-PCR (mRT-PCR) assay was developed for detection and differentiation of the Apple stempitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), whichare viruses frequently occurring in pear trees. Different combinations of mixed primer pairs were testedfor their specificity and sensitivity for the simultaneous detection of the three viruses. Three primer pairswere used to amplify their fragments of 247 bp, 358 bp and 500 bp, respectively. The primer pair for ASPVwas designed in this work, while the primer pairs for ACLSV and ASGV were from previous reports. Thesensitivity and specificity of the mRT-PCR assay for the three viruses were comparable to that of eachuniplex RT-PCR. The mRT-PCR was applied successfully for the detection of three viruses in leaves ofpear and apple plants, but was unreliable in the detection of ASGV in dormant barks. In conclusion, thismRT-PCR provides a useful tool for the routine and rapid detection and the differentiation of three pearviruses.