H3N2 influenza viruses not only cause seasonal epidemics in humans but also circulate widely in animals, posing a threat to both animal and human health. Our previous studies indicate that H3N2 avian influenza viruses (AIVs) are readily detected in live poultry markets (LPMs); however, the evolution and biological characteristics of the H3N2 viruses in poultry farms in China are unclear. In this study, we performed active surveillance and collected 49,135 samples from poultry farms. In total, 21 H3N2 AIVs were isolated and their genetics, receptor-binding properties, and replication in mice were evaluated. Phylogenetic analysis indicated that H3N2 AIVs harbor complicated gene constellations and have undergone extensive reassortment; the viruses derived their genes from 12 different hemagglutinin subtypes of duck viruses, including H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, and H14. The complicated gene constellations indicated that H3N2 viruses may have been introduced into poultry farms from different sources, but none have become dominant in poultry farms. Although the H3N2 AIVs possessed avian-type receptor-binding preference, most of the isolates could replicate without preadaptation and some of H3N2 viruses caused weight loss in mice. Notably, two H3N2 viruses acquired the PB2 627K mutation after a single round of replication in mice, suggesting similar mutations could occur if they replicated in humans. Overall, our study demonstrates that the H3N2 AIVs pose a potential threat to the public health and emphasizes the need for continued surveillance of H3N2 viruses in the both LPMs and poultry farms.