CD4+ Foxp3+ regulatory T-cells (Tregs) play a critical role in tolerance and immune regulation. Some studies propose that Tregs can lose the transcription factor Foxp3, their master regulator, and become so-called “ExTregs”. These studies suggest that ExTregs would lose both their identity and suppressive function, adopting conventional T-cell properties. However, the stability of the Treg lineage and the very existence of ExTregs in vivo is subject to much debate. This is in large part due to the absence of known markers to characterize ExTregs. Using a fate mapping system and significant technological improvements, we have unambiguously identified ExTregs in vivo in mice for the first time. The generation of ExTregs in lymphoid and non-lymphoid tissues was observed at steady-state in young and adult mice. Notably, the presence of ExTregs was found to be highly increased under inflammatory conditions in models of cancer but not auto-immune disease. Using multiparametric flow cytometry and scRNA-seq analyses, we revealed that ExTregs have their own signature that differs from Tregs and conventional T-cells. Our findings prove that ExTregs exist in vivo and have their own identity and functional properties. Our research introduces methodological improvements for the identification of ExTregs and will allow further investigations into the stability of Tregs.