H3N2 avian influenza virus (AIV) is prevalent in poultry and wild birds; however, certain strains possess the potential to evolve and cross the species barrier, thus posing a significant threat to public health. This study investigated the genetic evolution and pathogenicity of H3N2 subtype AIVs in southern China. The results showed that all eight gene segments of the nine isolates were clustered within the Eurasian lineage, with internal genes derived from multiple subtypes (including H1, H2, H3, H4, H5, H6, H7, and H9), indicating complex gene reassortment of H3N2 AIVs. Three representative isolates (LZD44, NND98, and NND100) replicated in various tissues of chickens and mice. Significantly, the LZD44 virus, which harbored the mammalian-adaptive mutations PB2-MVV and NP-I353V, exhibited higher virulence in chickens and mice than the other two strains. These findings indicate that H3N2 AIVs possess the capability to transmit to other species without prior adaptation, highlighting a risk of interspecies transmission. Therefore, continuous monitoring of H3N2 subtype AIVs is crucial for the effective prevention and management of avian influenza.