Highly pathogenic avian influenza (HPAI) H5N8 virus has caused considerable economic losses to poultry industry and poses a great threat to public health. Our previous study revealed two genetically similar HPAI H5N8 viruses displaying completely different virulence in mice. However, the molecular basis for viral pathogenicity to mammals remains unknown. Herein, we generated a series of reassortants between the two viruses and evaluated their virulence in mice. We demonstrated that 283M in PB2 is a new mammalian virulence marker for H5 viruses and that synergistic effect of amino acid residues 283M and 526R in PB2 is responsible for high virulence of the HPAI H5N8 virus. Analysis of available PB2 sequences showed that PB2 283M is highly conserved among influenza A viruses, while PB2 526R presents in most of human H3N2 and H5N1 isolates. Further study confirmed that the residues 283M and 526R had similar impacts on an HPAI H5N1 virus, suggesting that influenza viruses with both residues may replicate well in mammalian hosts. Together, these results present new insights for synergistic effect of 283M and 526R in PB2 of H5 HPAI virus on virulence to mammalian host, furthering our understanding of the pathogenesis of influenza A virus.