Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTLs) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared with equivalent numbers of more extensively expanded monoclonal CTLs, and are associated with complete remissions (CRs) in select patients. We applied high-throughput T cell receptor Vβ sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo after infusion, and then deduce the preadoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo posttransfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable with enumeration by binding of antigen peptide–human leukocyte antigen multimers, revealing that quantitative HTTCS is a reliable, multimer-independent alternative. The polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57 to 90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTLs enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative or survival advantages. Along with results indicating that naïve cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early differentiation phenotypes when selecting subset populations for adoptive transfer.