In recent years, H9N2 subtype avian influenza has become the dominant virus subtype in China´s chicken and duck populations. It is important to carry out long-term surveillance and pathogenic characteristics of the H9N2 virus. The embrocated eggs of specific pathogen-free chickens were used for virus isolation from clinical samples and the genomic RNA of H9N2 isolates was subjected to RT-PCR amplification, and then followed by sequencing analysis. Eight strains were isolated use the embryonated eggs of specific pathogen-free chickens. HA gene phylogenetic analysis indicated that h9.4.2.5-like H9N2 viruses were still have predominant during 2017～2021. Several specific amino acid mutations associated with mammalian adaptation were obtained, such as I155T, H183N, V190T, N198T, Q226L, Q227M, which increased its ability to spread across species. Additionally, six internal genes of H9N2 virus are clustered with the highly pathogenic H7N9 strain, the highly pathogenic H5N6 strain from Shandong Province, and the low pathogenic H3N8 and H10N8 viruses that have broken out in recent years, while the highly pathogenic H5N6 strains from Guangdong, Guangxi and Jiangsu do not form a branch with these strains. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2017-2021. The genetic evolution of the H9N2 avian influenza virus has endowed it with a stronger ability of cross-species infection, increasing the risk of the virus cross-species infecting humans and causing an outbreak. In addition, the genetic evolution and antigenic drift of the H9N2 virus may reduce the efficacy of existing vaccines. Researchers need to adjust the vaccine strain in a timely manner based on the genetic variations of the virus to enhance the specificity and effectiveness of the vaccine.