Abstract Background Japanese encephalitis virus (JEV) is a leading identified cause of encephalitis in Asia, often occurring in rural areas with poor access to laboratory diagnostics. We evaluated two rapid diagnostic tests (RDTs) for anti-JEV immunoglobulin M (IgM) detection. Methods Consecutive cerebrospinal fluid and serum from 388 patients (704 samples) with suspected JEV infections admitted to six hospitals in Laos were tested with one of two SD-Bioline anti-JEV IgM RDTs and the World Health Organization standard anti-JEV IgM enzyme-linked immunosorbent assay (ELISA; Panbio Japanese Encephalitis–Dengue IgM Combo ELISA. Results and Conclusions The performance of both RDTs showed strikingly low sensitivity in comparison to anti-JEV IgM antibody capture ELISA (2.1–51.4%), suggesting low sensitivity of the RDTs. We highlight the fundamental prerequisite to validate RDTs prior to use to ensure that they meet standards for testing.