Epitopes on the surface of the foot-and-mouth disease virus (FMDV) capsid have been identified by monoclonal antibody (mAb) escape mutant studies leading to the designation of four antigenic sites in serotype A FMDV. Previous work focused on viruses isolated mainly from Asia, Europe and Latin America. In this study we report prediction of epitopes in African serotype A FMDVs and tested selected epitopes using reverse genetics. Twenty-four capsid amino acid residues were predicted to be of antigenic significance by analyzing the capsid sequences (n=56) using in-silico methods and six residues by correlating capsid sequence with serum-virus neutralization data. The predicted residues were distributed on the surface-exposed capsid regions, VP1-VP3. The significance of residue changes at eight of the predicted epitopes was tested by site directed mutagenesis using a cDNA clone resulting in the generation of 12 mutant viruses involving seven sites. The effect of the amino acid substitutions on the antigenic nature of the virus was assessed by virus neutralisation (VN) test. Mutations at four different positions, namely VP1-43, VP1-45, VP2-191, and VP3-132 led to significant reduction in VN titre (P-value = 0.05, 0.05, 0.001 and 0.05, respectively). This is the first time that the antigenic region encompassing amino acids VP1-43 to 45 (equivalent to antigenic site 3 in serotype O), VP2-191 and VP3-132 were predicted as epitopes and serologically evaluated for serotype A FMD viruses. This identifies novel capsid epitopes of recently circulating serotype A FMD viruses in East Africa.