Almost 40% of the immune response directed towards mismatched HLA allografts in solid organ transplantation can be attributed to cross-reactive anti-viral memory T cells. These cells have the potential to mediate adverse clinical outcomes, with chronic DNA viruses including cytomegalovirus (CMV) contributing to post-transplant morbidity and mortality. We have identified CMV-reactive (HLA-A*02:01/NLVPMVATV) CD8+ T cells with a unique TCR signature (TRAV3TRAJ31_TRBV12-4TRBJ1-1; coined OTN5) that cross-react with specific HLA-B27 allomorphs in a defined hierarchy (B*27:07>B*27:09>B*27:05). The OTN5 TCR expressed on SKW3 cells (SKW3.OTN5) fails to recognise TAP-deficient T2 cells transfected with individual HLA-B27 allomorphs, highlighting the peptide dependence of the allorecognition. However the identity of the HLA-B27 restricted self-peptide(s) is unknown. Utilising an immunoproteomics approach for allopeptide identification, immunoaffinity chromatography was used to capture HLA-B27/peptide complexes from APC expressing either non-cross-reactive or cross-reactive allomorphs. The bound peptide ligands were isolated, fractionated by RP-HPLC and screened for immunogenicity against SKW3.OTN5 cells. A fraction capable of activating the OTN5 TCR was identified across all HLA-B27 allomorphs, but only in the context of the APCs expressing B*27:07 or B*27:09. Peptide sequences within this fraction and neighbouring non-immunogenic fractions were determined by mass spectrometry (LC-MS/MS) and a shortlist of peptides selected for functional screening based on peptide abundance. The data also indicate that a common B27 ligand(s) are differentially recognised by the alloreactive OTN5 TCR when in complex with different B27 allomorphs. This is consistent with our in depth analysis of micropolymorphic changes in other HLA supertypes where shared ligands are frequently presented in different conformations due to buried antigen-binding cleft polymorphisms. Moreover, whilst preliminary investigations have yet to identify the allopeptide(s) sequence, this study demonstrates the power of immunoproteomics in identifying naturally presented peptides in the context of specific HLA allomorphs in order to determine their role in mediating T cell responses.