Continuous monitoring and analysis of the evolution, recombination and epidemic of avian influenza virus will help to prevent and control the emergence of new avian influenza virus. The genes of 153 H5N6 avian influenza viruses selected from our long-term surveillance data and the GISAID for the 2016-2019 period were dissected in detail. Our analysis shows that the genes of H5N6 AIVs have been divided into more lineages, and that mutations leading to amino acid replacement of hemagglutinin and neuraminidase occurred mainly between 2017 and 2018. All H5N6 viruses are differentiated into at least 20 distinct genotypes derived from different evolutionary pathways. G1 genotype has replaced other genotypes to become dominant genotype of AIV. The results of animal experiments show that H5N6 of different genotypes has high virulence to mice. which indicates that H5N6 AIVs, especially the reassortment strains, are a potential threat to public health.