AbstractTo characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94‐, Tokyo/6/98‐, and Shiga/T30/98‐related lineage) of the Yamagata/16/88‐like lineage. The NS genes of the viruses belonging to the Harbin/7/94‐related lineage have additional three nucleotides at positions 439–447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88‐ and Victoria/2/87‐like lineages, but were closely related to that of the Yamagata/16/88‐like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87‐like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98‐like virus, and two strains of Harbin/7/94‐related lineage had acquired the various gene segments from Shiga/T30/98‐like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses. J. Med. Virol. 74:132–140, 2004. © 2004 Wiley‐Liss, Inc.