Abstract The antigenic relationship between Eurasian avian-like H1N1 swine influenza viruses (EA H1N1) and human pandemic 2009 H1N1 viruses (2009/H1N1) remains a critical question for influenza surveillance and vaccine efficacy . This study systematically investigated the antigenic differences between strains A/swine/Tianjin/312/2016 (TJ312, EA H1N1) and A/Guangdong-Maonan/SWL1536/2019 (GD1536, 2009/H1N1). Cross-hemagglutination inhibition (HI) assays revealed a significant antigenic disparity, with a 16-fold reduction in heterologous versus homologous HI titers . Comparative sequence analysis identified 22 amino acid differences across the five major antigenic sites (Sa, Sb, Ca1, Ca2, and Cb) of the HA1 subunit. Using reverse genetics , a panel of mutant viruses was generated. This study revealed that a single histidine (H)-to-asparagine (N) substitution at residue 128 (H3 numbering) in the Sa antigenic site acts as a primary determinant of antigenic variation , sufficient to ca...