Abstract To date, analysis of mice expressing TCR-β transgenes derived from CD4+ T cell clones has demonstrated equivalent or higher TCR diversity in naturally occurring regulatory CD4+ T cells (Tregs) versus conventional CD4+ T cells (Tcons). However, TCR-α–chain diversity in these mice may be influenced by the inherent bias toward the CD4+ lineage in the selected repertoires. We wished to determine whether the choice of TCR-β–chain influences the relative diversity of the Treg and Tcon repertoires, examining as a model the B6.2.16β-transgenic mouse, in which the fixed β-chain is derived from a CD8+ T cell clone. B6.2.16β Treg thymocytes showed significantly lower TRAV17 (AV9) CDR3 sequence diversity than both syngeneic Tcon thymocytes, and Treg and Tcon thymocytes from wild-type C57BL/6 (B6) mice. The ratio of single-positive CD4+/single-positive CD8+ thymocytes in B6.2.16β mice was similar to that in B6, yet both the proportional frequency and absolute number of CD4+Foxp3+ cells was significantly lower in the thymi and peripheral lymph nodes of B6.2.16β mice. Furthermore, B6 + B6.2.16β→B6 mixed bone marrow chimeras revealed that the transgenic β-chain disadvantaged Treg development in a competitive environment. These data underline the importance of the β-chain in assessments of Treg α-chain diversity and provide further support for the notion that interclonal competition for entry into the Treg lineage is a significant factor in determining the composition of this lineage.