Abstract Apple Chlorotic Leaf Spot Virus (ACLSV; family Betaflexiviridae, genus Trichovirus) and Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) are economically important viruses of apple (Malus × domestica Borkh.). A duplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of both ACLSV and ApMV along with an internal control (NADH dehydrogenase subunit 5 gene). The internal control was used to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of ApMV while for ACLSV & internal control previously reported primer sets were used. Firstly the uniplex RT-PCR assay was standardized for the detection of each virus independently. Then duplex RT-PCR protocol for simultaneous detection of ACLSV and ApMV was standardized by using primer pairs ACLSV-CP1F & ACLSV-CP1R and ApMV-CP1F & ApMV-CP5R, respectively, along with primer set for internal control. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both viruses in crab apples, rootstocks and different popular cultivars of apple. Results reveal that the duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. This optimized duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV & ApMV by reducing the time and cost of the supplies.