Abstract: Objective To understand the biological characteristics and genotype of Japanese encephalitis virus (JEV) strains newly isolated in Yunnan province. Methods The sequencing of full-length genomes was conducted for 4 strains of JEV isolated in Yunnan during 2009-2010. Clustal X, DNAstar, MEGA and other software was used to analyze the nucleotide (nt) data, deduced amino acid (aa) sequence and phylogenetic trees. Results Four JEV strains (YN09M57, DH10M865, DH10M978 and DHL10M62) were isolated from Culex tritaeniorhynchus samples in central and western Yunnan. The result of sequence analysis showed that the length of genomes of YN09M57 was 10 967 nt, encoding 3432 aa polyprotein, the lengths of genomes of DH10M865, DH10M978 and DHL10M62 were all 10 965 nt, encoding 3432 aa polyprotein. The 4 strains shared homology of genome ranging from 98.3% to 99.9% in nt and from 99.5% to 99.9% in aa. Compared with 7 JEV strains genotype Ⅰ in GenBank, the homology ranged from 98.3% to 99.9% in nt and from 99.7% to 99.9% in aa, and the homology with JEV genotype Ⅱ, Ⅲ, Ⅳ and Ⅴ in GenBank ranged from 78.7% to 89.7% in nt and from 91.4% to 98.4% in aa, respectively. There were 15 aa divergences which were not the key virulence loci in E protein compared with vaccine strain SA14-14-2. Phylogenetic analyses based on E and full-length genomes all showed that the 4 JEV strains from Yunnan belonged to genotype Ⅰ. The result also showed that 4 new JEV strains had close phylogenetic relationship with the strains from neighboring provinces and Southeast Asian. Conclusion This study indicated that JEV strains newly isolated in Yunnan were the members of JEV genotype Ⅰ. The key virulence loci and antigenic loci in E protein did not change. These results suggested that these strains had relatively stable genetic characteristics and distinct regional features.