Background H5N6 subtype avian influenza virus (AIV) continues to erupt in poultry, and it also threatens human public health safety. The goal is to clarify the evolutionary characteristics of the 3 strains of H5N6 subtype AIV and the variation of special amino acid sites, and to clarify the pathogenic characteristics of the virus to chickens, ducks and mice.3 strains of H5N6 subtype AIV named as A/ Environment/ Fujian/ ENIVER109/ 2020 (H5N6) , A/ Duck/ Fujian/ DK039/ 2020 (H5N6) , A/ Duck/ Fujian/ DK153/ 2020 (H5N6) . The genetic evolution and variation of the 3 viruses were analyzed by whole genome sequencing. Chickens, ducks and mice were infected with 3 strains of viruses through the nasal cavity, and the pathogenic ability and pathogenic characteristics of the viruses to different animals were observed. Results The HA genes of all three viruses are in the 2.3.4.4h clade. The ancestor of the DK039 strain may have been different from the other two viruses. Mutations with cross-species transmission and enhanced pathogenicity were found in all three viruses. Infected chickens, ducks and mice showed differences in body weight, clinical signs, apparent and microscopic lesions in organs, and viral titers in organs. Conclusions The isolated H5N6 subtype AIV has many mutations associated with pathogenicity and cross-host transmission, and the pathogenicity of the virus varies from animal to animal.