H3 subtype avian influenza viruses (AIVs) are frequently detected in poultry and wild birds, however, systematic characterization of contemporary isolates remains limited. We aimed to investigate the genetic evolution, pathogenicity, and transmission characteristics of H3 subtype AIVs circulating in Eastern China. Seven H3 subtype AIVs isolated between 2014 and 2021, including five H3N2, one H3N3, and one H3N6 strain, were analyzed. Phylogenetic analysis showed that all isolates belonged to the Eurasian lineage. Evidence of extensive reassortment with other AIV subtypes, as well as adaptive mutations associated with pathogenicity, and cross-species transmission, particularly in H3N2 subtype AIVs, was identified. Notably, H3N2 subtype AIVs exhibited dual receptor-binding properties, recognizing both SA α-2,3-Gal and SA α-2,6-Gal receptors. Although all isolates demonstrated low pathogenicity in chickens, mice, and guinea pigs, variations in transmission efficiency were observed. The H3N2 strain A/Duck/Anhui/LY/2021 showed the highest capacity for cross-species and aerosol transmission among guinea pigs. Overall, these findings indicate that H3 subtype AIVs have the potential for cross-species transmission and highlight the importance of continued surveillance of H3 subtype AIVs circulating in nature.