Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance.