The high prevalence of influenza A virus is identified in Hunan Province because of the high density of poultry farms. To survey the variations of H9N2 subtype avian influenza virus in Hunan province, we analyzed HA and NA genes of 10 virus strains isolated from different areas of Hunan Province. All these strains belong to the Eurasian lineage, Y280-like sub-lineage. The cleavage sites in their HA genes were all RSSR↓GLT, corresponding to the feature of low pathogenic AIV. All strains had an L (Leu) at the site 234 in the HA genes, indicating the ability of binding with the SAα-2,6 receptor. NA gene stalk deletions at aa 63–65 were also detected from all the isolates, indicating a possibility of increased virus replication in mammals. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.