Background and Objectives After allogeneic haematopoietic stem cell transplantation (SCT) the whole T-cell receptor (TCR) repertoire shows a markedly skewed pattern for 2–3 years. A small fraction of CD4+ T cells is represented by CD25+ regulatory lymphocytes (T reg), which play a crucial role in modulating peripheral tolerance. To investigate their ability to react to the massive antigenic stimulation generated in an allogeneic host, which could significantly affect their pattern of reconstitution, we analyzed the TCR repertoire of T reg after SCT, focusing on the degree of similarity to CD4+ CD25− conventional T cells (T conv). Design and Methods We assessed the TCR Vβ repertoire of T reg in ten patients who had received allogeneic SCT, by using complementarity determining region 3 (CDR3) spectratyping. We developed a new similarity score for the analysis. This score expresses the proportion of Vβ with similar profile between T reg and T conv. Results For up to 3 years after SCT the repertoires of T reg and T conv were characterized by several Vβ with different profiles between the two cell subsets, while they were extremely similar in patients more than 3 years post-allografting (similarity score= 0.90 vs. 0.61). The differences observed early after SCT were mainly ascribable to Vβ expressing an oligoclonal profile in T conv but not in T reg. Interpretation and Conclusions Our data show that the TCR repertoires of T reg and T conv are significantly different early post-SCT, while they tend to become identical with full reconstitution. This difference could reflect either a discrepancy in the in vivo reactivity against common antigenic stimulations or be the result of different post-transplant ontogeny.