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How TCRV-region bias impacts TCR\u03b1\u03b2 heterodimer selection and resultant diversity is unclear. The D(b)PA(224)-specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) \u03b2-length of 6 aa. Despite these restrictions, D(b)PA(224)-specific BV29(+) T cells use a wide array of unique CDR3\u03b2 sequences. Structural characterization of a single, TRBV29(+)D(b)P(A224)-specific TCR\u03b1\u03b2-pMHCI complex demonstrated that CDR3\u03b1 amino acid side chains made specific peptide interactions, but the CDR3\u03b2 main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in V\u03b2-region use. In support of this hypothesis, retrovirus expression of the D(b)PA(224)-specific TCRV\u03b1-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. 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