Primer-extension sequencing of the RNA template of polio, dengue, Rift Valley fever, and Japanese encephalitis (JE) viruses has provided new information on their geographic distribution, origin, and evolution. In a previous study of 46 diverse JE virus strains, we demonstrated the existence of three distinct JE genotypes in Asia. We now report the occurrence of a fourth genotype. In the present study, 19 JE virus isolates, representing various geographic regions of Asia and a 50-year time span, were compared with each other and with Murray Valley encephalitis, West Nile, and Kunjin viruses. Twelve of the JE strains from the Indonesian Archipelago and the Philippines had not been previously examined; the remainder were representatives of the three previously identified genotypes. Two hundred forty nucleotides from the pre-M gene region of the virus were used in these comparisons. Using 12% divergence as a cut-off point, the 19 JE strains fell into four distinct genotypic groups; maximum divergence across the comparison region was 21%. The newly recognized fourth genotype was comprised of five Indonesian isolates that were 7% divergent from the rest of the JE viruses.