H9N2 subtype avian influenza virus (AIV) is widespread globally, with China being the main epidemic center. Inactivated virus vaccination was adopted as the main prevention method in China. In this study, 22 hemagglutinin (HA) sequences were obtained from all inactivated vaccine strains of H9N2 subtype AIVs in China since its introduction. A phylogenetic analysis of the vaccine sequences and HA sequences of all published H9N2 subtype AIVs was conducted to investigate the relationship between vaccine use and the virus genetic diversity of the virus. We found that during 2002-2006, when fewer vaccines were used, annual genetic differences between the HA sequences were mainly distributed between 0.025 and 0.075 and were mainly caused by point mutations. From 2009 to 2013, more vaccines were used, and the genetic distance between sequences was about 10 times greater than between 2002 and 2006, especially in 2013. In addition to the accumulation of point mutations, insertion mutations may be the main reason for the large genetic differences between sequences from 2009 to 2013. These findings suggest that the use of inactivated vaccines affected point mutations in the HA sequences and that the contribution of high-frequency replacement vaccine strains to the rate of virus evolution is greater than that of low-frequency replacement vaccine strains. The selection pressure of the vaccine antibody plays a certain role in regulating the variation of HA sequences in H9N2 subtype AIV.