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H9N2 subtype avian influenza virus (AIV) was the highly contagious pathogen which has caused severe losses in the poultry industry throughout China in recent years. Using current epidemic viruses as vaccine was an effective way to prevent infection of H9N2 subtype AIV. In this study, a total of 23 H9N2 subtype AIV strains were isolated in 200 samples from 13 provinces of China during 2012-2016. The sequencing and phylogenetic analysis of the hemagglutinin gene sequence of the isolation strains showed that 22 isolation strains were clustered to h22.214.171.124 lineage, while only 1 belonged to h126.96.36.199. The data of cross-HI, neutralization and cross-immune protection shown that the A/chicken/Hunan/HN/2015 (HN) and A/chicken/Shandong/SD/2014 (SD) strains as vaccine could effectively protect present viruses infection compared with other strains. These results indicated that current epidemic viruses were mainly belong to h188.8.131.52 lineage and HN and SD strains as candidate vaccine strains were potentiality for the protection of present H9N2 subtype AIV infection.