Abstract: Objective To study the genetic characterization of human enterovirus 71 (EV-71) in Chenzhou City, Hunan Province during 2010 to 2011, and provide scientific data for HFMD control and prevention. Methods 60 anal swab specimens sent by each county (city, district) of Chenzhou were selected randomly for virus isolation, Realtime RT-PCR was used for detecting enterovirus RNA. Specimens of 14 strains (Four strains of them were isolated at the summer epidemic peak in 2010, and the others were mainly isolated at the winter epidemic peak in 2011) with cytopathic and EV71 RNA positive were amplified by RT-PCR, and then nucleotide sequencing was performed and aphylogenetic tree was constructed using bioinformatics method. Results 14 EV71 strains were clustered with C4a subgenotype reference strains (EU703812) of C genotype in phylogenetic tree. The 14 selected strains showed high homology (96.1% - 100.0% and 99.3% - 100.0%) in nucleotide and translated nucleotide homology analysis, and higher nucleotide homology (91.8% - 93.3%) and amino acid homology (99.3%) with the Fuyang isolated strains (C4a subgeno type reference strain, EU703812) than A, B, and C subgeno type reference strains K98E, S283T, and A293S were found between Chenzhou isolated strains and Fuyang isolated strains (EU703812). The significant difference of nucleotideor amino acid sequence was not found between HEV71 strains from severe (dead) patients and mild patients. Conclusion EV71 was the predominant pathogen that caused HFMD in Chenzhou City in 2010 and 2011, which belonged to C4a genotype. High homology and stability were found in the 14 isolated EV71 strains of Chenzhou. No amino acid mutations were found to be associated with the case type.