High variability of influenza B virus (IBV) hemagglutinin (HA) impairs the cross- neutralization ability of vaccines, leading to reduce efficacy. We identified significant differences in cross-neutralization between IBV strains B/Wyoming/06/2014 and B/Brisbane/60/2008, which differ in only three amino acid residues. The 214 T point mutation was found to dramatically enhance cross-neutralization (>
10-fold). Antibody-based reverse validation also revealed that this mutation significantly increased the neutralization capacity (500-62,500-fold). Furthermore, monitoring revealed that the mutation rate at this site has reached its highest level in nearly 20 years, with a prevalence exceeding 80% in sequences submitted from certain regions. Our findings provide new evidence for the selection of vaccine strains with improved cross- neutralization effects, which will aid the development of broad-spectrum vaccines by modifying minimal antigenic epitopes.