Regulatory T cells (T_regs) constitute a fascinating subpopulation of CD4⁺ T cells due to their ability to limit the immune response against self and non-self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for T_reg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of T_reg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of T_regs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which T_regs derive.