Avian influenza virus (H9N2) infection is a major problem of product performance in poultry worldwide. Vaccination is used to limit spread, but more knowledge is needed on the epidemiology of virus subtypes to improve vaccine design. In this study, 40 H9N2 subtype avian influenza viruses (AIVs) were isolated from vaccinated poultry flocks in China from 2010 to 2011. Hemagglutinin (HA) from different virus strains was sequenced and analyzed. We found that the HA genes of these strains shared nucleotide and deduced amino acid homologies that ranged from 90.1 to 92.9 and 91.4 to 95.0 %, respectively, when compared with vaccine strains. Phylogenetic analysis showed that the strains tested could be divided into two major groups. Group I consisted of 24 strains isolated mainly from Eastern and Central China. Group II consisted of 20 strains isolated from Southern China. The cleavage site within the HA protein contained two basic motifs, PSRSSR↓GLF for group I, and PARSSR↓GLF for group II. Additional potential glycosylation sites were found at amino acid position 295 in the HA1 of the isolates in group I, compared with isolates in group II and the vaccine strains. Furthermore, 38 out of the 40 isolates had a leucine residue at position 216 (aa 226 in H3), which was characteristic of human influenza virus-like receptor specificity. In the present study we found that geographical factors play a significant role in virus evolution, and emphasize the importance of continuing surveillance of H9N2 AIVs in chickens in China.