After serial passage of a waterfowl-origin H3N2 subtype avian influenza virus in BALB/c mice, we obtained H3N2 mouse-adapted variants and identified eight amino acid substitutions in five viral proteins in our previous study. Here, we analyze the key mutations determining viral pathogenicity in mammals. We found that both PB2-D701?N mutation and M1-M192?V mutation were implicated in the viral pathogenic phenotypic variation of H3N2 avian influenza virus in mice. Furthermore, we found that PB2-D701?N could enhance viral replication in vitro and in vivo and expanded viral tissue tropism. Our data suggest that PB2-D701?N and M1-M192?V are the virulence markers of H3N2 avian influenza virus, and these markers can be used in the trans-species transmission surveillance for the H3N2 avian influenza virus.