PURPOSE Post-transplant immune monitoring remains limited by a lack of tools to identify donor-specific T cells involved in graft rejection. Our previous work led to the discovery of immunodominant donor peptide-MHC (pMHC) epitopes that account for a large proportion of allo-responses. The aim of this study was to develop pMHC multimer tools incorporating these epitopes to detect and phenotype donor-specific T cells during transplant immune responses. METHODOLOGY BALB/c (H-2d) recipient mice received heart transplants from C57BL/6 (H-2b) donors (total n=18 donor-recipient pairs). Heart-infiltrating leukocytes and splenocytes were examined at different time points post-transplant. The cells were stained with a 12-plex pMHC multimer panel tagged with DNA barcodes and a syngeneic multimer as a control. Single-cell analysis was performed to determine full-length TCR sequences, transcriptomes, and pMHC specificity. RESULTS The multimer panel identified a substantial proportion of donor-specific T cells in rejecting heart grafts compared to the negative control (20.3±1.1% vs 0.4±0.1%, p<0.001). The proportion of donor-specific T cells from the spleens was much higher on day 7 post-transplant compared to their pre-transplant stage (3.6±0.3% vs 1.3±0.2%, p<0.005). Single-cell analysis revealed distinct phenotypes and clonal expansion of donor-specific T cells during graft rejection. CONCLUSION This study represents a significant advance by enabling precise monitoring of donor-specific T cells. The findings have translational potential for improving transplant immune response monitoring, optimising immunosuppressive therapies, and facilitating the development of tolerance-inducing protocols in clinical transplantation.