Abstract CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Vα24-negative and 13 canonical Vα24-positive/Vβ11-positive human NKT cell clones generated using α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. The Vα24-negative clones expressed Vβ11 paired with Vα10, Vα2, or Vα3. Strikingly, their Vα-chains had highly conserved rearrangements to Jα18, resulting in CDR3α loop sequences that are nearly identical to those of canonical TCRs. Vα24-positive and Vα24-negative clones responded similarly to α-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3α loop is sufficient for recognition of α-GalCer despite CDR1α and CDR2α sequence variation. Unlike Vα24-positive clones, the Vα24-negative clones responded poorly to a glucose-linked glycolipid (α-glucosylceramide), which correlated with their lack of a conserved CDR1α amino acid motif, suggesting that fine specificity for α-linked glycosphingolipids is influenced by Vα-encoded TCR regions. Vα24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jα18-positive TCRs that can recognize α-GalCer. One α-GalCer-reactive, Vα24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR β-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR α-chain elements contribute to α-linked glycosphingolipid specificity, whereas TCR β-chains can confer heterogeneous additional reactivities.