During local respiratory disease outbreaks, occurring in 2003 and 2004 in horse training stables within race-tracks in Rome, and on a stud horse farm in Bari in 2005, four strains of equine influenza (EI) virus were isolated. All outbreaks occurred in flu-vaccinated horses. Here, we are reporting the results of the genetic characterization of these isolates, together with that of another EI virus strain isolated in 1999 from a dead foal presenting pulmonary lesions. Alignment and phylogenetic analyses were carried out using the haemagglutinin amino acid sequences. The Rome and Bari isolates were identified as members of the American lineage, closely related to other recent strains isolated in America as well as in Europe, including the latest recommended American lineage vaccine prototype A/eq/SouthAfrica/4/2003. In contrast, the Italian 1999 isolate was clustered within the European lineage. In Italy, the most recent outbreaks of EI have been caused by the currently circulating American-like strains, even in vaccinated populations, confirming that vaccines should contain an updated representative strain of this lineage. Presently, companies are still in the process of registering updated vaccines but no product is yet available on the market.