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This study was performed to assess the seroprevalence against Japanese encephalitis virus (JEV) in chickens collected from an urban area of Denpasar and a rural area of Karangasem in Bali. A total of 142 domestic chickens were sampled and then tested using enzyme linked immunosorbent assay (ELISA) to detect the antibodies. The seroprevalence was calculated in each of the clustered areas and the seropositivity was associated with age and sex of the sampled chickens. The results showed that chickens collected in Denpasar had 97.10% (n=70, 95% CI: 90.8899.52) seroprevalence against JEV, whereas the chickens collected from Karangasem Regency had a seroprevalence of 93.05% (n=72, 95% CI: 85.2897.41). In association with area collection, age and sex, chickens collected from Denpasar were 2.5 times (OR: 2.5, 95% CI: 0.4813.53, P>0.05) more likely to be seropositive containing the antibodies against JEV compared to the chickens from Karangasem Regency. Mean-while, chickens of age equal to or more than six months were 6.2 times (OR: 6.2, 95% CI: 1.1533.07, P0.05) more likely to be seropositive compared to females. No significant difference was observed for the seroprevalence in the chickens collected in both areas even though it was found to be significantly associated with the chickens’ age.