The continued diversification of the H9N2 avian influenza virus (AIV) into multiple antigenically and phylogenetically distinct lineages is promoting the emergence of strains with pandemic potential. Constant monitoring of the genetic evolution and changes in biological characteristics of the H9N2 viruses is therefore essential. In this study, we analyzed the genetic evolution of the H9N2 viruses isolated from poultry farms between 2022 and 2023 and evaluated their pathogenicity in chickens and mice. The HA genes of all ten isolates belonged to the h9.4.2.5 lineage, which is currently the predominant evolutionary lineage in China. Yet, their HA genes further divided into distinct subbranches within the h9.4.2.5 lineage. The NA genes of these viruses shared high homology with the prevalent H9N2 AIVs in recent years. However, these viruses were located in different evolutionary groups. Notably, the internal genes showed close relationships with those of recent H3, H6, and H9 subtype AIVs, suggesting active reassortment events among co-circulating viruses. Pathogenicity assessment in mice and chickens demonstrated divergent virulence between two representative isolates, FS22 and JM14, which clustered into different h9.4.2.5 subbranches. FS22 exhibited more efficient and prolonged replication in the lungs and turbinates of mice compared to JM14. Both viruses replicated efficiently in the lungs, kidneys, and trachea of chickens at 3 days post-infection (DPI), but differed in their horizontal transmission potential. Particularly, inoculated and contacted chickens all produced high antibody levels from the 5 DPI until the end of the experiment, and peak antibody titers for both viruses occurred at 7 DPI. These findings underscored the continuous evolution ofH9N2 AIV enhanced its genetic and phenotypic diversity, leading it to pose a threat to public health. Thus, continuous surveillance in poultry farms is necessary.