T-cells are key players in the establishment of the adaptive immune response. T-cells are classified into two lineages, CD4⁺ or CD8⁺ T-cells, based on the expression of either CD4 or CD8 co-receptors. Interestingly, accumulating evidence supports the existence of a third T-cell lineage; mature CD4⁺CD8⁺ double-positive T-cells (DPs). Mature peripheral DPs are detectable in a variety of lymphoid and non-lymphoid tissues (spleen, lymph nodes, blood, and gut), species (human, mouse, pig, rat, and monkey), and pathological scenarios such as cancer (melanoma, T-cell lymphoma), suggesting that DPs might affect the outcome of autoimmune and malignant skin disorders. The study of this cell-subset presents several difficulties: peripheral-DP are a minority within the large T-cell compartment, and 98% of un-sorted cells that appear to be DPs are in fact CD4⁺-CD8⁺ T-cell aggregates. We have overcome these issues using a isolation/enrichment strategy which allows us to analyze a >95% pure DPs suspension. To determine whether naïve peripheral DPs arise from CD4⁺ T-cells or CD8⁺ T-cells, we analyzed the T-cell receptor (TCR) diversity by flow cytometry using monoclonal antibodies that recognize the variant region of the β chain (TCRVβ). Interestingly, principal component analysis (PCA) of the surface expression of 14 TCRVβ chains in individual cells demonstrated that DPs from the spleen clustered with CD4⁺ T-cells from the spleen and not with CD4⁺ T-cells from lymph nodes. Moreover, the high proportion of Vβ7 and Vβ6-expression by DP cells in the lymph-nodes was sufficient to differentiate this population from the rest. Finally, DP-thymocytes were an intermediate population between single-positive CD8⁺ and CD4⁺ T-cells as biologically reported. Our results demonstrate that peripheral DPs do not originate from the direct seeding of immature DP-thymocytes to the periphery, but from tissue-specific CD4⁺ T-cells, which, after colonization of the organ, re-express CD8αβ potentially conferring functional changes.