The H3 avian influenza virus (AIV) is a common avian pathogen, widely prevalent among domestic poultry and wild birds. In China, H3N8 AIVs led to two confirmed cases of human infection in 2022, followed by a deadly case in 2023. In 2019, an H3N8 AIV was identified in chickens monitored within live poultry markets of eastern China. To investigate the genetic connections between these eastern Chinese AIV strains and other influenza viruses, we sequenced all gene segments of these strains and performed a comparative analysis with sequences retrieved from the GenBank database. We performed phylogenetic analysis and molecular characterization of the virus and then assessed the viral growth kinetics and pathogenicity in mice. The results demonstrated that the H3N8 AIV possesses genetic features of H3, H4, H7, and H10 AIVs, which have been identified in ducks and chickens in China. The strain could stably replicate in DF-1, MDCK, and A549 cells. In animal experiments, the strain demonstrated moderate pathogenicity and replicated in mice without prior adaptation. In conclusion, a reassortment occurred between AIVs from diverse avian hosts, and the ongoing dissemination of H3N8 AIVs may pose a threat to human health.