AbstractInfluenza B virus hemagglutinin (HA) is a major surface glycoprotein with frequent amino acid substitutions. However, the roles of antibody selection in the amino acid substitutions of HA were still poorly understood. In order to gain insights into this important issue, an analysis was conducted on a total of 271 HA1 sequences of influenza B virus strains isolated during 1940–2007. In this analysis, phylogenetic analysis by maximum likelihood (PAML) package was used to detect the existence of positive selection and to identify positively selected sites on HA1. Strikingly, all the positively selected sites were located in the four major epitopes (120‐loop, 150‐loop, 160‐loop, and 190‐helix) of HA identified in previous studies, thus supporting a predominant role of antibody selection in HA evolution. Of particular significance is the involvement of the 120‐loop in positive selection, which may become increasingly important in future field isolates. Despite the absence of different subtypes, influenza B virus HA continued to evolve into new sublineages, within which the four major epitopes were targeted selectively in positive selection. Thus, any newly emerging strains need to be placed in the context of their evolutionary history in order to understand and predict their epidemic potential. J. Med. Virol. 81:114–124, 2009. © 2008 Wiley‐Liss, Inc.