In order to explore Japanese encephalitis virus carried by mosquitoes in farms of Hunan Miluo, a JEV pathogenic investigation on mosquitoes was conducted in three farms of the region. Gene JEV NS5 collected from about 3 900 mosquitoes in 3 pig farms was amplified using RT–PCR. The result showed that out of 17 in total 40 samples was positive. The JEV was isolated from the positive grinding fluid using the BHK–21 cell method. One JEV was obtained after 3 passages, and was named by HNML1. A pair of primer was designed according to gene JEV E, gene E from HNML1 was amplified, and sequencing and homology analysis were conducted as well. The results showed that the homology on nucleotide sequence and amino acid sequence of gene E to corresponding sequences in live antigen attenuated vaccine strains SA14–14–2 was 86.9% and 97.0%, respectively. There were 15 differences of amino acids, which included 8 key amino acids related to virulence. The result from gene homology analysis showed that HNML1 was a type strain of JEV I.