Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3β robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the β-chain variable region (V_β) family present in the TCR or the length of the CDR3β. An index based on these findings distinguished V_β2⁺, V_β6⁺ and V_β8.2⁺ regulatory T cells from conventional T cells and also distinguished CD4⁺ T cells selected by the major histocompatibility complex (MHC) class II molecule I-A^g7 (associated with the development of type 1 diabetes in NOD mice) from those selected by a non–autoimmunity-promoting MHC class II molecule I-A^b. Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.