The D225G (aspartic acid to glycine) substitution in the hemagglutinin of H1N1 influenza virus may alter its receptor-binding specificity. Direct analysis of polymorphisms in 126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N1 2009 virus from 117 clinical specimens in Hong Kong found the D225G substitution for 7 (12.5%) of 57 patients with severe disease and for 0 (0%) of 60 patients with mild disease. D225G quasispecies were identified mainly in endotracheal aspirate samples and were identified less frequently in nasopharyngeal aspirate samples from patients with severe disease. Continuous monitoring of the prevalence and tissue tropism of this variant during its circulation among humans is important.