To identify the pathogen of acute hemorrhagic conjunctivitis (AHC) in Shandong Province in 2010, eye mucous swab samples were collected from 26 patients in Qingdao and Linyi City. Real time-PCR assays for EV70, CVA24 and Adenovirus were performed on these samples. The result showed 17 samples (65.39%) were CVA24 positive while all the samples for HEV70 and Adenovirus detection were negative, which implied that CVA24 was the causative pathogen of this outbreak. A total of 10 virus strains isolated on Hep-2 cells were identified as CVA24 through VP1 amplification and nucleotide sequence analysis. The nucleotide and amino acid homologies on VP1 region among these isolates were 99.3%-100.0% and 99.5%-100.0%, respectively, and the strains aggregated together to one clade in phylogenetic tree. These results showed that the CVA24 circulating in Qingdao and Linyi City belonged to one transmission chain. Shandong CVA24s segregated into 5 different clades, and great nucleotide divergence was observed be tween AHC isolates and others.