The cross-reactivity of Japanese encephalitis virus (JEV)-immunized chicken sera and West Nile virus (WNV)-immunized chicken sera in serological tests, such as the IgG indirect ELISA (IgG-ELISA), hemagglutination inhibition test (HI) and plaque reduction neutralization test (PRNT), for JEV and WNV were examined. The mean JEV/WNV ELISA ratio in IgG-ELISA of JEV-immunized sera was significantly higher than that of WNV-immunized sera (P<0.01). JEV-immunized chicken sera did not cross-react in the WNV HI. However, all the WNV-immunized chicken sera cross-reacted in the JEV HI. JEV-immunized chicken sera did not show the WNV neutralization titer at 90% plaque reduction, and WNV-immunized chicken sera did not showed the JEV neutralization titer at 90% plaque reduction. Therefore, it is possible that chicken JEV serum can be distinguished from WNV serum by comparing the titers of IgG-ELISA, HI or PRNT respectively.