Apple, Malus domestica Borkh, in the Rosaceae family is a fruit tree compatible with cool climate. Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) type members of Capilovirus , Foveavirus and Trichovirus , respectively in the family Betafexiviridae are the major causal agents of viral diseases of apple trees. These viruses are usually symptomless on trees and cause over 60 percent reduction of apple fruit production particularly, in mixed infections. Development of rapid, accurate and sensitive detection methods is essential to make correct management decisions for control of viral diseases. The aim of this study was to optimize a multiplex RT-PCR for simultaneous detection of ASGV, ASPV and ACLSV in apple trees. For this purpose, sampling was done in the different seasons of 2013 and 2014 from Isfahan, Lorestan, Chaharmahal va Bakhtiari and Markazi provinces.‌ Total RNA was extracted from leaves and blossoms of apple trees and subjected to RT-PCR method using specific primer pairs including ASGV-F/ASGV-R for ASGV (713 bp), ASPA/ASPC for ASPV (264 bp) and A52/A53 for ACLSV (358bp). The expected size bands were obtained in RT-PCR assays. The amplicons were sequenced and BLAST search confirmed the infection of the samples with ASGV, ASPV and ACLSV. Simultaneous detection of ASGV, ASPV and ACLSV was performed using duplex RT-PCR assay with binary combinations of specific primer pairs and then using multiplex RT-PCR with three primer pairs (ASGV-F/ASGV-R, ASPA/ASPC and A52/A53). The results indicated the multiplex RT-PCR method is an efficient method for simultaneous detection of ASGV, ASPV and ACLSV. One hundred and ten collected samples were tested with multiplex RT-PCR method. The results showed 70, 53 and 35 samples were infected with ASGV, ASPV and ACLSV, respectively. In addition, the mixed infection of viruses was identified 37%, 24%, 19% and 16% for ASGV+ASPV, ACLSV+ASGV, ACLSV+ASPV and ACLSV+ASGV+ASPV, respectively. To our knowledge, this is the first use of multiplex RT-PCR method for simultaneous detection of viral infections of apple trees in Iran. According to the results, multiplex RT-PCR is a sensitive, accurate, affordable and reliable method for detection of apple tree viruses. Keywords : Apple stem grooving virus , Apple stem pitting virus , Apple chlorotic leaf spot virus , Multiplex RT-PCR, Mixed Infection.