Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, the focussing of the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterisation of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHCI homozygous animals respectively. In this study, peptide-MHCI tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T-cells. During validation of these tetramers a substantial proportion of Tp249-59-reactive T-cells failed to bind the tetramer, suggesting this population was heterogeneous with respect to the recognised epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross reactivity. The Tp249-59 and Tp250-59 epitopes utilise different serine residues as the N-terminal anchor for binding to the presenting MHCI molecule. Molecular dynamic modelling predicts that the 2 peptide-MHCI complexes adopt structurally different conformations and TRB sequence analysis show that Tp249-59 and Tp250-59 are recognised by non-overlapping TCR repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for TCR recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant.