We previously revealed that Japanese encephalitis virus (JEV) seroprevalence was 4·5% in pigs on Ishigaki Island from 2005 to 2007. However, a partial E gene sequence (151 bp) of the JEV genome (JEV/sw/Ishigaki/1/2005) was detected in one pig. Phylogenetic analysis showed that JEV/sw/Ishigaki/1/2005 belonged to genotype III and to the same lineages isolated in Taiwan from 2006 to 2008. Serum samples were collected from 128 pigs on Ishigaki from 2009 to 2010, 24 wild boars on Ishigaki from 2008 to 2010, and 117 wild boars on Iriomote Island from 2008 to 2010. Four (3·1%) pigs on Ishigaki were positive for JEV antibody, but all wild boars on the island were negative. Fifty-two (44·4%) wild boars on Iriomote were positive for JEV antibody, in contrast to a seroprevalence of 3·7% in 2000 and 2004. JEV on Iriomote and/or in Taiwan might be related to transmission on Ishigaki.