In April 2004 an outbreak of equine influenza occurred at the Zagreb hippodrome, Croatia. Clinical respiratory disease of the same intensity was recorded in vaccinated and non-vaccinated horses. The equine influenza vaccine used in Croatia at the time of the outbreak contained the strains A/equine/Miami/63 (H3N8), A/equine/Fontainebleau/79 (H3N8) and A/equine/Prague/56 (H7N7). At the same time, the usual strains in vaccines used in Europe were, in accordance with the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel on equine influenza, A/equine/Newmarket/1/93 (H3N8) and A/equine/Newmarket/2/93 (H3N8). At the same time, some current vaccines in the USA contained A/equine/Kentucky/97 (H3N8). Genetic characterization of the HA1 portion of the haemagglutinin (HA) gene of virus isolated from the outbreak indicated that the isolate (A/equine/Zagreb/04) was an H3N8 strain closely related to recent representative viruses of the American lineage Florida sub-lineage. In comparison with both H3N8 vaccine strains used in horses at the Zagreb hippodrome, A/equine/Zagreb/04 displayed amino acids changes localised to 4 of the 5 described antigenic sites (A-D) of subunit protein HA1. Comparison of the amino acid sequence of the HA1 subunit protein of the outbreak strain with that of A/equine/Newmarket/1/93 displayed three amino acids changes localised in antigenic sites B and C, while antigenic sites A, D and E were unchanged. The Zagreb 2004 outbreak strain had the same amino acids at antigenic sites of the HA1 subunit protein as the strain A/equine/Kentucky/97. Amino acid changes in antigenic sites between HA1 subunit of the outbreak strain and the strains used in the vaccines likely accounted for the vaccine failure and the same clinical signs in vaccinated and unvaccinated horses. Use of a recent strain in vaccines should limit future outbreaks.