Abstract: Objective To detect West Nile virus(WNV)genome and isolate Japanese encephalitis virus(JEV)from the mosquitoes collected during the epidemic season of Japanese encephalitis in Nanyang of Henan province. Methods Mosquito samples were collected in 2006 to detect WNV genome by nest PCR and isolate viruses by inoculation onto C6/36 and BHK-21cells. The JEV isolates were identified by serological and molecular biological methods, and the PrM and E gene were amplified. The sequence alignment and phylogenetic analysis of JEVs were carried out by using biological software. Results A total of 6231 mosquito samples were collected and 7 strains of virus were isolated by observing CPE inoculated on cells. All the isolates were JEV, and no WNV positive sample was found by nest PCR. The 7 JEV strains belonged to genotype I both by using PrM and E gene sequences. Among the isolates, the homology of 98.5%-100% in nucleotide sequence and 98.4%-100% in amino acid sequence were found in E gene. Compared with the vaccine strain P3 and SA14-14-2, the nucleotide sequence differences were 87.7%-88.2% and 87.3%-87.9% respectively. In the deduced important amino acid sites that affect the virulence, all of the newly isolates showed the same to P3 strain but not to SA14-14-2 strain. Conclusion Genotype I was the only type of the JEVs isolated from mosquitoes in Nanyang. There were some differences between the isolates and the vaccine strains in E gene. But these variations were not in the important sites that affect the virulence of JEV. No WNV positive sample was detected by nest-PCR.