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Background: Thailand has introduced a nationwide vaccination against Japanese encephalitis virus (JEV) into National Immunization Programme since the 1990's. To improve the understanding of immunity and susceptibility of the population after 28 years of a vaccination programme, we conducted a JEV seroepidemiological study in a JEV-endemic area of Thailand., Methods: An age-stratified, population-based, seroepidemiological study was conducted in Chiang Mai, Thailand-a northern Thai province where is an endemic area of Japanese encephalitis. Nine districts were chosen based on administrative definition: rural (n = 3); urban (n = 3); and peri-urban (n = 3). Within each district, eligible participants were randomly selected from 3 age groups: adolescents (10-20 years); adults (21-50 years); and older adults/elderly (≥51 years) by computer randomization. Plaque reduction neutralization tests (PRNT50 and PRNT90) were performed to measure neutralizing antibodies to JEV. To account for the cross-reactivity of JEV and other flaviviruses, JEV seroprotection was defined according to age, previous history of JEV vaccination, and PRNT50/PRNT90 levels of study participants., Results: Overall, 279 adolescents, 297 adults, and 297 older adults/elderly were enrolled from nine districts. Age-stratified, protocol-defined, cluster-adjusted JEV seroprotection rates were 61% (95% CI: 48-73%), 43% (95% CI: 31-57%), and 52% (95% CI: 37-67%) for adolescents, adults, and older adults/elderly, respectively. Living in peri-urban districts, having a history of prior dengue virus infection, and previously receiving mouse brain-derived JEV vaccine were significantly associated with seroprotection to JEV in adolescents. Older age and male sex were associated with seroprotection for adults; and only male sex was the associated factor for older adults/elderly (P <0.05)., Conclusions: Approximately half of population living in a JEV-endemic area demonstrated seroprotection to JEV. Ongoing nationwide surveillance on JEV seropepidemiology is an important strategy to understand the evolving population-level immunity to JEV, and to help formulating the appropriate recommendations on JE immunization., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.F.T., P.A., R.H., and J.N. work or worked for Sanofi, a vaccine company which produces Japanese encephalitis and other vaccines, and may hold shares and/or stock options in the company for the Sanofi employees. All other authors declare no conflicts of interest related to this study.