Japanese encephalitis virus (JEV) is the most commonly identified cause of acute encephalitis syndrome (AES) in Asia. The WHO recommended test is anti-JEV IgM-antibody-capture-enzyme-linked-immunosorbent-assay (JEV MAC-ELISA). However, data suggest this has low positive predictive value, with false positives related to other Flavivirus infections and vaccination. JEV RT-PCR in cerebrospinal fluid (CSF) and/or serum is highly specific, but is rarely positive; 0–25% of patients that fulfil the WHO definition of JE (clinical Acute Encephalitis Syndrome (AES) and JEV MAC-ELISA positive). Testing other body fluids by JEV RT-qPCR may improve the diagnosis. As a pilot study thirty patients admitted to Mahosot Hospital 2014–2017, recruited to the SouthEast -Asia-Encephalitis study, were tested by JEV MAC-ELISA and two JEV real-time RT-PCR (RT-qPCR) assays (NS2A and NS3). Eleven (36.7%) were JEV MAC-ELISA positive. Available CSF and serum samples of these patients were JEV RT-qPCR negative but 2 (7%) had JEV RNA detected in their throat swabs. JEV RNA was confirmed by re-testing, and sequencing of RT-qPCR products. As the first apparent report of JEV RNA detection in human throat samples, the provides new perspectives on human JEV infection, potentially informing improving JEV detection. We suggest that testing patients' throat swabs for JEV RNA is performed, in combination with molecular and serological CSF and serum investigations, on a larger scale to investigate the epidemiology of the presence of JEV in human throats. Throat swabs are an easy and non-invasive tool that could be rolled out to a wider population to improve knowledge of JEV molecular epidemiology. Evidence continues to implicate Japanese encephalitis virus (JEV) as a major cause of encephalitis in Asia, with recent evidence of possible autochthonous transmission in Africa 1–3. Among the key factors in its persistent role as a public health problem are the limitations of existing diagnostic tests, our understanding of its epidemiology and inadequate implementation of vaccination programmes. The conventional mainstay of JEV encephalitis diagnosis is serology, with serum and cerebrospinal fluid (CSF) anti-JEV IgM antibody capture enzyme-linked immunosorbent assays (JEV MAC-ELISA) recommended by the World Health Organization (WHO) 4. However, there are concerns about the accuracy of JEV MAC-ELISA for diagnosing JEV. Low positive predictive value of JEV MAC-ELISA has been reported 5 , and there are recognised difficulties with false positives related to vaccination and in areas where other Flavivirus infections are endemic 6,7. Diagnosis of JEV by Reverse-Transcription (RT)-PCR is highly specific, and enables improved understanding of the molecular epidemiology of JEV 8. However, JEV RT-PCR testing of CSF and serum samples has low sensitivity , rarely positive in patients at presentation (0–25% that fulfil the WHO definition of Acute Encephalitis