Forty-seven strains of H9 subtype avian influenza viruses identified by specific reverse transcription-PCR method were isolated from the chicken and duck flocks in different areas of China during the 2002 to 2009 epizootic period. Hemagglutinin (HA) genes of these strains were sequenced and analyzed with the representative strains published in GenBank. The results indicated that the HA genes of these strains and the vaccine strains displayed nucleotide homologies ranging from 91.7 to 96.6% and amino acid homologies ranging from 92.3 to 95.7%, respectively. Analysis of the mature peptide sequences of these HA genes showed that the presence of leucine at position 216 (corresponding to residue 226 in H3 numbering) indicated a preference to the binding of alpha (2-6) sialic acid receptors, which was the same as human isolates. Extra potential glycosylation sites appeared in the HA genes of most tested isolations compared with the vaccine strains. The HA cleavage sites of most of the strains were the 335PSRSSR downward arrow GLF341, but all of the strains met the characteristics of low-pathogenic avian influenza. The results of phylogenetic analysis indicated that all 47 strains and the current vaccine strains belong to the same phylogenetic lineage h9.4.2, but they had some genetic deviation in the last decade. Compared with the vaccine strains, 7 mutations were found in the antigen epitope region of the HA genes of the field strains. These results suggested that the commercial vaccine might not induce satisfactory prevention against infection of H9N2 avian influenza virus.