In this study, a PCR-DGGE protocol was standardized in order to distinguish Victoria and Yamagata influenza B lineages directly from clinical samples. After routine multiplex PCR characterization, amplicons of the haemagglutinin gene bearing a 40bp-length GC clamp were generated by nested-PCR and analyzed by electrophoresis in 6% polyacrylamide gel with a 25-45% urea-formamide gradient. The results showed a perfect correlation between DGGE and phylogenetic analyses for all compared samples, besides some distinct profiles in Victoria and Yamagata groups that could be used to infer variability inside these groups. In summary, this DGGE protocol for the haemagglutinin gene is rapid, useful and efficient, being an alternative for discrimination between the influenza B lineages.