Tissue-resident memory T (TRM) cells are a non-circulating lymphocyte population that are principally located in peripheral tissues. TRM cells provide rapid protection against a wide range of infections and cancer; hence, enhancing TRM cell formation and persistence is an attractive means for establishing durable immunity. While many studies have dissected the properties of TRM cells within peripheral tissues in mice, our knowledge of human T cells has been largely derived from blood sampling. In collaboration with Austin Health, we have established the first Australian Donation and Transplantation Biobank that provides access to a wide range of healthy human organs. Using this resource, we performed a whole-body analysis of TRM cells across barrier and non-barrier tissue sites. We employed multiparameter flow cytometry and scRNAseq to resolve distinct TRM cell populations across the gut, skin, liver and spleen. We observed intra- and inter-organ TRM cell heterogeneity based on the expression of tissue residency markers CD69 and CD103, and inhibitory molecules such as PD-1 and CD244. Furthermore, we have demonstrated how the tissue microenvironment influences various TRM cell functional capabilities. Together, this holistic characterisation of TRM cells across solid organs underscores the importance of investigating local tissue immunity which cannot be discovered by conventional blood sampling. The results of this study will direct novel tissue-specific immunotherapies aimed to promote and establish durable tissue immunity.