The H6N6 avian influenza virus (AIV) subtype is one of the most frequently isolated subtypes in poultry, and it has a broad host range. Some strains can overcome species barriers for transmission and infect humans. Increased affinity for human-type receptors is a key factor in this process. In this study, two H6N6 AIV strains originating from five different clades, in which amino acid 226 of hemagglutinin was mutated from glutamine to lysine, were isolated from ducks. The receptor-binding preference and pathogenicity of the two strains in poultry and mice were evaluated. The results indicated that the DK/GD/W3 strain can bind to both α-2,6 and α-2,3 receptors, whereas the DK/GD/L31 strain maintained affinity toward avian-origin α-2,3 receptors, highlighting differences in receptor tropism and pathogenicity to different hosts for two H6N6 strains with the same genetic background. These findings have revealed the complex recombination characteristics and molecular characteristics of H6N6 circulating strains in the environment and underscored the importance of continuous surveillance of this subtype for livestock and poultry health as well as human safety.