To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the Nterminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RTPCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor 1α (EF1 α). This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.