An outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Yunnan Province, China between August and September in 2007. A total of 3,597 cases were officially reported and the incidence rate reached 1390.94/100,000. Descriptive epidemiological analysis of the outbreak was conducted using the data from National Disease Supervision Information Management System (NDSIMS). To determine the causative agent for this outbreak and to analyze their genetic features, 30 conjunctival swabs and 19 paired serum specimens of acute and convalescent phase were collected from 30 patients with AHC, and viral isolation, molecular typing, antibody assay and phylogenetic analysis were performed. 11 virus strains were isolated from 30 conjunctival swabs. Amplification and sequencing of the VP4 region of these strains identified that coxsackievirus A24 variant (CA24v) could be the causative agent of the AHC outbreak and this was further confirmed by subsequent virus neutralizing antibody test on 19 paired serum specimens. Phylogenetic analysis based on the 3C regions showed that the Yunnan CA24v strains belonged to Group 3 and clustered with the strains isolated from worldwide AHC outbreaks after 2002. Phylogenetic analysis based on the partial VP1 revealed that the Yunnan strains differed from the strains isolated from AHC outbreak in Guangdong of China in 2007 with 2.8 - 3.0% nucleotide divergence, suggesting that two different lineages of CA24v caused the independent AHC outbreaks in Yunnan and Guangdong, respectively.