Globally more than 160,000 organ/tissue transplants are performed per annum, most with genetic mismatches between the human leukocyte antigen (HLA; human MHC) molecules carried by the donor and recipient. HLA mismatches can trigger destructive immune responses leading to graft rejection and loss. Recipient T cells that mediate these immune responses recognise foreign donor HLA molecules in complex with self-peptides (1). A major limitation following transplantation has been an inability to identify the peptide/HLA (pHLA) targets of graft rejection and the alloreactive T cells that directly recognise them. To determine the role of the self-immunopeptidome in direct allorecognition we utilised a mouse skin transplant model mismatched for H2-Kb (2). Different tissues from both donor (spleen, skin) and recipient (hepatocytes) were collected for immunopeptidome mapping involving sequential immunoaffinity purification for capture and dissociation of peptide/H2-Kb complexes, and RP-HPLC for peptide fractionation. Reconstituted peptides were analysed by LC-MS/MS using an information-dependent acquisition strategy on a Q-Exactive Plus Hybrid Quadrupole Orbitrap, with peptide identities assigned using PEAKS Studio X+ at 5% FDR. A total of 8,752 peptides were identified across all three tissues, with 1083 being shared. We selected 100 shared peptides for functional screening to determine their immunogenicity and observed that 17 were recognised by more than 5% of the alloreactive CD8+ T cells. Importantly, as few as 5 different specificities collectively identified ~40% of alloreactive T cells demonstrating that there are immunodominant allogeneic peptide/MHC complexes that account for a large proportion of the anti-transplant response. The findings of this study represent a significant advance in our understanding of the role of endogenous peptides in direct T cell alloreactivity, and underpins future development of diagnostic reagents for monitoring of post-transplant alloreactive T cells in humans.