H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs´ infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.