Three strains of H6N6 subtype avian influenza virus (AIV) were isolated from live-poultry markets of central China during 2009-2010. A phylogenetic analysis showed that these isolates originated from gene reassortment among different virus lineages of the H6 subtype. In an experimental infection of animals, the selected isolate was non-pathogenic for chickens and low-pathogenic for mice. The wild-type isolate was capable of replication in mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaption in mouse lung. The genomes of viruses of passage 0 (P0), P4, and P8 were sequenced and compared, and virulence-related amino acid substitutions were found in multiple sites during mouse lung passage.