The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Curculionidae), which is native to North America, is an invasive wood-boring insect species in China with disastrous consequences. Plant quarantine is an important method to hinder the invasion of this species. In order to distinguish RTB from the other four closely related bark beetles, Scolytus schevyrewi Semenov, Xyleborus artecomans Schedl, Xyleborus interjectus Blandford and Blastophagus minor Hartig, which are frequently intercepted at Chinese ports, we developed a nested polymerase chain reaction (PCR) assay. The specificity of the nested PCR primers to recognize RTB was confirmed by the failure to amplify genomic DNA from the other four Scolytinae species. The sensitivity was approximately 40 pg of a recombinant RTB plasmid. We evaluated the validity of the assay by analyzing bark beetles intercepted at quarantine stations. Our findings demonstrate that this specific molecular method allows for the rapid identification of D. valens for pest risk assessment and plant quarantine.