Avian influenza viruses (AIVs) are naturally preserved in waterfowls and sometimes spill over to infect humans and mammalian animals. The H3 subtype is one of the most prevalent influenza virus subtypes in waterfowls. Since 2000, H3 subtype AIVs have been continuously isolated from poultry and wild birds in the mainland of China, which implied a dynamic spread in large-scale geography and multiple species. Combinations of H3 with N1–N8 subtypes were reported, among which H3N2 and H3N8 subtypes predominated. Frequent mutations and reassortments increased the genetic diversity associated with altered virus virulence, transmission, and mammalian adaptation of H3 AIVs, posing a potential threat to animal and human health. This study systematically analyzed the epidemiology, genetic characteristics, and mammalian adaptation-related mutations of H3 subtype AIVs in the mainland of China, facilitating the H3 subtype AIVs research and risk assessment.