Following solid organ transplantation, peptide presentation by human leukocyte antigen (HLA) molecules on the cell surface of donor tissues enables peptide-dependent recognition by recipient alloreactive T cells, ultimately driving immunological destruction and graft rejection. Yet, the precise alloreactive T cell populations involved in graft rejection, as well as the peptide-HLA (pHLA) complexes they engage, remain largely uncharacterised. This lack of insight hinders progress toward clinical assays and therapies capable of identifying and treating early rejection. We hypothesise that recipient alloreactive CD8⁺ T cells recognise a subset of shared donor self-peptides expressed on transplantable tissues. This study characterised the HLA-A*02:01 (A2) immunopeptidome to define which peptides trigger alloreactive CD8⁺ T cell responses in primary tissues from organ donors. Here, pHLA complexes were isolated from heart, lung, liver and kidney samples using immunoaffinity isolation of HLA class-I molecules, with peptides subsequently separated by RP-HPLC and identified by mass spectrometry. In total, 171,563 HLA-A2-restricted peptides were identified across 18 distinct tissues from 11 organ donors. Among these, 282 shared 9mer peptides were present in all tissues. The top 57 peptides were subsequently screened for their ability to activate T cells. Our findings reveal that anti-HLA-A2 alloreactive CD8⁺ T cell lines from two individuals (R10 and R20) each exhibited focused specificity by recognising only a single epitope, peptide 57 for R10 and peptide 45 for R20. Currently we are working on identifying the exact sequences of the alloreactive CD8+ T cell receptors however, this work opens exciting avenues for development of more specialised post-transplant immune-monitoring strategies.