Translating studies on T cell function and modulation from mouse models to humans requires extrapolating in vivo results on mouse T cell responses in lymphoid organs (spleen and lymph nodes [LN]) to human peripheral blood T cells. However, our understanding of T cell responses in human lymphoid sites and their relation to peripheral blood remains sparse. In this study, we used a unique human tissue resource to study human T cells in different anatomical compartments within individual donors and identify a subset of memory CD8(+) T cells in LN, which maintain a distinct differentiation and functional profile compared with memory CD8(+) T cells in blood, spleen, bone marrow, and lungs. Whole-transcriptome and high-dimensional cytometry by time-of-flight profiling reveals that LN memory CD8(+) T cells express signatures of quiescence and self-renewal compared with corresponding populations in blood, spleen, bone marrow, and lung. LN memory T cells exhibit a distinct transcriptional signature, including expression of stem cell-associated transcription factors TCF-1 and LEF-1, T follicular helper cell markers CXCR5 and CXCR4, and reduced expression of effector molecules. LN memory T cells display high homology to a subset of mouse CD8(+) T cells identified in chronic infection models that respond to checkpoint blockade immunotherapy. Functionally, human LN memory T cells exhibit increased proliferation to TCR-mediated stimulation and maintain higher TCR clonal diversity compared with memory T cells from blood and other sites. These findings establish human LN as reservoirs for memory T cells with high capacities for expansion and diverse recognition and important targets for immunotherapies.