AbstractSince the 2017 Southern Hemisphere influenza season, the A(H1N1)pdm09-like virus recommended for use in the vaccine was changed because human, but not ferret, sera distinguish A(H1N1)pdm09 viruses isolated after 2013 from the previously circulating strains. An amino acid substitution, lysine to glutamine, at position 166 (H3 numbering) in the major antigenic site of HA was reported to be responsible for the antigenic drift. Here, we obtained two anti-A(H1N1)pdm09 HA monoclonal antibodies that failed to neutralize viruses isolated after 2013 from a vaccinated volunteer. Escape mutations were identified at position 129, 165, or 166 in the major antigenic site of HA. Competitive growth of the escape mutant viruses with the wild-type virus revealed that some escape mutants possessing an amino acid substitution other than K166Q showed superior growth to that of the wild-type virus. These results suggest that in addition to the K166Q mutation that occurred in epidemic strains, other HA mutations can confer resistance to antibodies that recognize the K166 area, leading to emergence of epidemic strains with such mutations.