A 280-nucleotide sequence from the capsid-premembrane (C/preM) gene region of 44 Japanese encephalitis (JE) virus strains isolated in Taiwan from mosquitoes from 1983 to 1994, and 3 strains, (Ling , Chang , and HV1 ) isolated from human brain were analyzed by direct sequencing of reverse transcription-polymerase chain reaction (RT-PCR) amplified products and compared with the corresponding sequences of reference strains. The overall sequence homology of the 47 isolates was > or = 93.3%. Taking 12% nucleotide divergence as a cut-off value, all isolates fell into genotype 3, which included strains from Japan, China, the Philippines, Sri Lanka, India, and Nepal. High nucleotide homology was observed among isolates from different regions of Taiwan and different time periods; on the other hand, high variation existed among isolates from the same region and time period. Phylogenetic analysis showed that the 47 Taiwan isolates fell into three clusters. Twenty-five isolates formed cluster 1, 18 isolates cluster 2, and four isolates cluster 3. Isolates in cluster 1 showed greater (< or = 2.9%) intragroup divergence compared to those in cluster 2 (< or = 1.1%) or cluster 3 (< or = 0.7%). The majority of isolates from northern (73.3%) and central (60%) Taiwan belonged to cluster 1, whereas most isolates (66.7%) from southern Taiwan belonged to cluster 2. Comparison with other Asian JE virus strains showed that isolates of cluster 1 were more specific to Taiwan than isolates of cluster 2 and cluster 3.