Several studies have identified residues apparently under positive selection in influenza B virus hemagglutinin. Host immune evasion is the main mechanism proposed to exert this selection pressure. However, these reports have not considered the culture history of the strains used to calculate positive selection. This article shows that passage of influenza B virus through egg culture is a strong contributory factor to the strength and statistical significance of positive selection on hemagglutinin. Non-synonymous mutations resulting in the loss of the N-glycosylation site at positions 197-199 of hemagglutinin have been positively selected to a far greater degree in egg-cultured strains than in other strains. Once egg-cultured strains are removed from the analysis, positive selection is found to be far weaker, less statistically significant, and more diffusely localized along the protein. Caution should therefore be exercised both in claims for the existence of positive selection in influenza B hemagglutinin, and in attribution of host immune evasion as its cause. The major cause of molecular adaptation in influenza B hemagglutinin proteins may well be laboratory eggs rather than natural hosts. J. Med. Virol. 82:123127, 2010. (C) 2009 Wiley-Liss, Inc.