The H3 subtype avian influenza virus (AIV) poses a threat to both animal and human health. In this study, phylogenetic analysis showed that the H3 AIVs had various genomic constellations and extensive reassortments, increasing genetic diversity and the emergence of new pathogenic viruses that might infect human beings. Molecular analysis demonstrated that the major molecular markers linked to drug resistance were identified in M genes of three studied viruses, and there might be wide range of resistant virus infections in poultry in the future. Although all the H3 viruses preferentially bound to the avian-type receptor, the growth kinetics experiments showed that the selected H3 viruses were capable of efficient replication in mammalian cells, suggesting a potential cross-species transmission of H3 viruses. Overall, our results emphasize the need for continued surveillance of H3 outbreaks and may also help us improve knowledge on H3 AIVs prevention and control.