The major viruses infecting apple throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV) and recently a new virus apple necrotic mosaic virus (ApNMV) was reported from mosaic affected apple cultivars in India. The aim of the present study was to detect the ApNMV along with 3 other viruses (ApMV, ASPV and ASGV) simultaneously through multiplex RT-PCR. Four primer pairs produced amplicon of 670bp, 550bp, 350bp, and 210bp corresponding to ApNMV, ApMV ASPV and ASGV respectively were found specific for these viruses tested individually. Annealing temperature (55 ºC), primer concentration (0.8ul) and other components of master mix were standardized for development of one step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated on 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of 1 to 4 viruses in these cultivars. Most of the viruses were found present either alone or in mixed infection, however, ASPV was more common in tested cultivars. Easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect 4 viruses infecting apple plants either in mixed infection or individually in the field. This assay will help in surveys, indexing of apple germplasm and distribution of all four viruses in apple growing regions of India.