We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicity in mice. We found that four amino acid mutations (PA-P224S, PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. Particularly, PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced its 50% mouse lethal dose (LD₅₀) by almost 1,000-fold.