The influenza B virus hemagglutinin contains four major antigenic sites (the 120 loop, the 150 loop, the 160 loop and the 190 helix) within the head domain. These immunodominant antigenic sites are the main targets of neutralizing antibodies, and are subject to antigenic drift. Yet, little is known about the specific antibody responses towards each site in terms of antibody prevalence and hemagglutination inhibition activity. Here, we used modified hemagglutinins of influenza B virus, which display only one or none of the major antigenic sites to measure antibody responses towards the classical as well as the non-canonical epitopes in mice, ferrets and humans. With our novel reagents, we found that both hemagglutination inhibition antibodies and total IgGs were mostly induced by the major antigenic sites. However, in human adults, we observed high hemagglutination inhibition antibody responses towards the non-canonical epitopes. By stratifying the human samples into age groups, the non-canonical antibody responses appeared to increase with age.IMPORTANCE This study dissects the specific antibody responses towards the major antigenic sites and the non-canonical epitopes of influenza B virus hemagglutinin in animals and humans using novel reagents. These findings will guide the design of the next generation influenza virus vaccines.