To investigate the seroepidemiology and genetic characterization of hepatitis E virus (HEV) in western Yunnan Province. Questionnaire survey was conducted among 1638 residents in western Yunnan Province using stratified sampling method. Enzyme-linked immunosorbent assay was used to detect serum anti-HEV IgG and IgM. HEV RNA was extracted from patients with serum anti-HEV IgM positive. The open reading flame 2 (ORF2) of HEV that was amplified by nested RT-PCR was sequenced and compared with standard HEV genotypes 1-4. Serum anti-HEV positive was found in 13.92% (228/1638) residents. The HEV infection rate in males was significantly higher than that in females with a ratio of 1.47 (P<0.01). 20-30 and 30-40 years old young men showed the highest incidence, 20.57% and 20.78%, respectively. While 10-20 and 20-30 years old young women exhibited the highest infection rate, 11.85% and 15.60%, respectively. According to occupation, the highest HEV infection rate was observed in farmers (20.35%) and migrants (16.50%). We isolated 10 individual HEV isolates from 31 patients with serum anti-HEV IgM positive. Homology analysis and phylogenetic analysis indicated that these 10 HEV isolates belonged to HEV genotype 4 with the homology of 78.65%-94.71%. The HEV infection rate is high in western Yunnan Province. HEV genotype 4 is the leading cause of HEV infection and young farmers and migrants are the main infected population.