The current circulating influenza B viruses can be divided into two major phylogenetic lineages: the Victoria and Yamagata lineages. We conducted a survey of influenza B viruses in Hubei and Zhejiang provinces during 2009-2010. Out of 341 throat swabs, 18 influenza B viruses were isolated. Five isolates were selected for genetic and phylogenetic analysis. The molecular analyses revealed that all the isolates had similar antigenic characteristics to B/Brisbane/60/2008. However, in the three viruses isolated from Zhejiang, a single asparagine to aspartic acid substitution in position 197 was observed, thereby eliminating the glycosylation at that site and possibly causing an antigenic change. None of the viruses had amino acid mutations at positions 116, 149, 152, 198, 222, 250, 291, and 402 of the neuraminidase (NA) gene, predicting that the viruses would still be sensitive to NA inhibitors. Phylogenetic analyses revealed that all five isolates were closely related to B/Brisbane/60/2008-the 2010 vaccine strain-and contained Victoria-like hemagglutinin and Yamagata-like NA genes, suggesting that reassortment may had occurred. In addition, similar phylogenetic patterns among the acidic polymerase, nucleoprotein and matrix protein genes, as well as between the basic polymerase 1 and basic polymerase 2 genes, were observed, suggesting possible functional interactions among these proteins. All the results highlighted the importance of molecular monitoring of influenza B viruses for reassortment and antigenic drift.