TheCulex vishnuisubgroup includes three important vectors of Japanese encephalitis virus,Culex tritaeniorhynchusGiles,Cx. pseudovishnuiColless, andCx. vishnuiTheobald, all of which occur in the Ryukyu Archipelago, Japan. Although these three species have been shown to be vectors of JE virus in many areas of Southeast Asia, it is not yet known what role each plays in the transmission of the virus in this region. Reliable identification of adult, field-collected specimens is a critical component in epidemiological studies of virus transmission. Mosquitoes in theCx. vishnuisubgroup can be reliably identified in the larval stage. However, because females of these species are very similar, it is difficult to distinguish among them using morphology. We developed a polymerase chain reaction (PCR) assay for the identification of these species. Three species-specific primers were developed for the PCR assay based on a comparative analysis of the nucleotide sequence of the first internal transcribed spacer (ITS1) in the ribosomal DNA gene array. The primers, CT2REV, CP1REV, and CV1REV were designed to amplify a single DNA fragment each fromCx. tritaeniorhynchus,Cx. pseudovishnui,andCx. vishnui,respectively, when paired with a single forward primer that is complementary to the highly conserved 18S rDNA gene. The amplified fragments were separated easily and identified on an agarose gel to facilitate species identification.