The potato rot nematode (Ditylenchus destructor) species is an influential pathogenic potato nematode. Many countries impose quarantine restrictions on the presence of D. destructor in imported plant and soil materials. Therefore, the present investigation was conducted from 2018 to 2020 at the All-Russian Plant Quarantine Center of Russia Federation with samples collected from the Severtsov Institute of Ecology and Evolution, RAS, Russia and Iran. In the current study, we developed PCR with species-specific primers for the rapid and reliable separation of D. destructor using gel electrophoresis and melting curve analysis. A species-specific primer set was designed based on the nucleotide sequence of the gene. The PCR protocol was verified using samples of D. destructor and the closely related species, D. dipsaci and D. gigas. The species-specific primer set was able to detect D. destructor from samples containing mixtures of Ditylenchus species. The PCR species-specific protocol should allow for more rapid identification of Ditylenchus species recovered from plant materials than previously possible. © 2020, Gaurav Society of Agricultural Research Information Centre. All rights reserved.