Equine influenza is a major cause of respiratory infections in horses and causes widespread epidemics, despite the availability of commercial vaccines. Antigenic drift within the haemagglutinin (HA) glycoprotein is thought to play a part in vaccination breakdown. Here, we carried out a detailed investigation of the 1989 UK outbreak, using reverse genetics and site-directed mutagenesis, to determine the individual contribution of amino acid substitutions within HA. Mutations at positions 159, 189 and 227 all altered antigenicity, as measured by haemagglutination-inhibition assays. We also compared HA sequences for epidemic and vaccine strains from four epidemics and found that at least 8 amino acid differences were present, affecting multiple antigenic sites. Substitutions within antigenic site B and at least one other were associated with each outbreak, we also identified changes in loop regions close to antigenic sites that have not previously been highlighted for human H3 influenza viruses.