Abstract The evolution of influenza B viruses is poorly understood. The viral mechanisms of escape from immunological pressure include insertion–deletion in the hemagglutinin (HA) and co-circulation of different lineages, but this last epidemiological picture was observed only in SE Asia in the last decade. Virological surveillance conducted in Italy during the 2001/2002 and 2002/2003 seasons and molecular and antigenic characterization of 107 isolates allowed to detect the co-circulation of strains belonging to the Victoria and Yamagata lineage during the former season. Yamagata-like strains belonged to the two sub-lineages, B/Harbin/7/94 and B/Sichuan/379/99, while Victoria-like strains were B/Hong Kong/330/01-like viruses. During the 2002/2003 season, all isolates belonged to the B/Shangdong/7/97 cluster. In conclusion, during the last two seasons, viruses belonging to the two lineages and the four sub-lineages were isolated. In consideration of the distinct HI test pattern of the two lineages and amino acid changes over the antigenic sites inter- and intra-sub-lineage, these findings could play an important role in choosing the composition of the vaccine and in the evaluation of its efficacy.