Regulatory T (Treg) cells are critical mediators of anti-tumor immunity and can influence response to immune checkpoint blockade (ICB). The functional heterogeneity of tumor-infiltrating Tregs (TIL-Tregs), particularly, the relative contributions of thymus-derived natural Tregs (nTregs) versus peripherally induced Tregs (iTregs) remain unclear. This limitation is largely due to the lack of robust discriminatory markers in human tumors. Here, we developed a murine tumor model to derive gene signatures that distinguish nTregs from iTregs within the tumor microenvironment (TME). Application of these signatures to murine tumors revealed transcriptionally distinct TIL-Treg populations, with minimal overlap between cells identified by each signature, indicating discrete nTreg and iTreg programs. Next, we applied these murine signatures to single-cell data from human TIL-Tregs treated with ICB and the gene signatures delineated cell populations across different cell types indicating heterogeneity and functionally distinct subsets. Interestingly, the iTreg gene signature preferentially identified cells in one of three “activated” TIL-Treg transcriptionally defined clusters that was significantly enriched in immunotherapy-treated tumors compared to untreated tumors. These findings suggest that iTregs are induced or selectively enriched upon ICB treatment. Ongoing work will further elucidate functional roles of nTregs and iTregs in the TME and their impact on response to ICB treatment.