A specific polymerase chain reaction assay was developed for direct identification of the distinct subgroup of plum pox potyvirus (PPV) isolates from cherry trees (PPV-cherry, PPV-C) and its differentiation from other known subgroups of PPV. The specificity of the assay is based on using a pair of primers whose nucleotide sequences are located on the coat protein gene of PPV-sour cherry (SoC) at regions of high nucleotide divergence between PPV-SoC and other isolates of PPV. The technique will be useful for studying the epidemiology of PPV-C as well as for practical testing in certification and quarantine programs worldwide.