Mouse type I natural killer T cell receptors (iNKT TCRs) use a single Vα14-Jα18 sequence and Vβs that are almost always Vβ8.2, Vβ7, or Vβ2, although the basis of this differential usage is unclear. We showed that the Vβ bias occurred as a consequence of the CDR2β loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent Vβ-CD1d affinities are further modulated by the hypervariable CDR3β loop, thereby defining a functional interplay between the two iNKT TCR CDRβ loops. These Vβ biases revealed a broadly hierarchical response in which Vβ8.2 > Vβ7 > Vβ2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.