H5N6 subtype avian influenza virus (AIV) continues to erupt in poultry, and there have also been many incidents of human infection. Its outbreak is devastating to the poultry breeding industry, and it also threatens human public health safety. Forty-five strains of H5N6 subtype AIV isolated from sick ducks in East China were used in this study. HA gene analysis found that the amino acid sequences at the cleavage site of the HA gene of the 45 isolates were all continuous basic amino acids, which was in line with the characteristics of highly pathogenic avian influenza. Amino acid analysis showed that specific amino acid substitutions and deletions occurred in both HA and NA proteins, indicating that 45 isolates may have the ability to infect mammals and humans. The results of the Muscovy duck (Carina moschata) infection experiment showed that the clinical symptoms and visceral pathologic changes of Muscovy ducks infected with the virus strains isolated in different years were similar. Mutations at multiple amino acid sites were found in the HA and NA genes of duck-derived H5N6 subtype AIV that were clinically isolated in Eastern China in our study. And the influence of some mutations on the pathogenicity and transmission ability of the virus is still unclear. Therefore, it is necessary to continuously monitor and study the H5N6 subtype AIV of ducks.