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The H9N2 subtype avian influenza viruses (AIVs) have been circulating in China for more than 20 years, attracting more and more attention due to the potential threat of them. At present, vaccination is a common prevention and control strategy in poultry farms, but as virus antigenicity evolves, the immune protection efficiency of vaccines has constantly been challenged. In this study, we downloaded the hemagglutinin (HA) protein sequences of the H9N2 subtype AIVs from 1994 to 2019 in China—with a total of 5138 sequences. The above sequences were analyzed in terms of time and space, and it was found that h9.4.2.5 was the most popular in various regions of China. Furthermore, the prevalence of H9N2 subtype AIVs in China around 2006 was different. The domestic epidemic branch was relatively diversified from 1994 to 2006. After 2006, the epidemic branch each year was h9.4.2.5. We compared the sequences around 2006 as a whole and screened out 15 different amino acid positions. Based on the HA protein of A/chicken/Guangxi/55/2005 (GX55), the abovementioned amino acid mutations were completed. According to the 12-plasmid reverse genetic system, the rescue of the mutant virus was completed using A/PuertoRico/8/1934 (H1N1) (PR8) as the backbone. The cross hemagglutination inhibition test showed that these mutant sites could transform the parental strain from the old to the new antigenic region. Animal experiments indicated that the mutant virus provided significant protection against the virus from the new antigenic region. This study revealed the antigenic evolution of H9N2 subtype AIVs in China. At the same time, it provided an experimental basis for the development of new vaccines. [ABSTRACT FROM AUTHOR]