γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t -distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV ⁺ and CMV ⁻ donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.