Mucosal-associated invariant T (MAIT) cells are highly conserved innate-like T cells in mammals recognized for their high baseline frequency in human blood and cytotoxic effector functions during infectious diseases, autoimmunity, and cancer. While the majority of these cells express a conserved CD8ab+ TRAV1-2 T cell receptor (TCR) recognizing microbially-derived Vitamin B2 intermediates presented by the evolutionarily conserved major histocompatibility complex I-related molecule, MR1, there is an emerging appreciation for diverse MAIT cell subsets that possess distinct functions including CD4+ MAIT cells that remain underexplored. In this study, we adopted an unbiased single-cell TCR-sequencing approach in MR1-5-OP-RU-tetramer-reactive T cells. We discovered that CD4+ MAIT cells are enriched with highly diverse TRAV1-2 negative TCRs. To specifically characterize this TCR repertoire, we analyzed VDJ sequences across two datasets and identified distinct TCR usage among CD4+ MAIT cells including TRAV21, TRAV8 (TRAV8-1, TRAV8-2, TRAV8-3), and TRAV12 families (TRAV12-2, TRAV12-3), as well as more variable J chain and CDR3 sequences. Non-TRAV1-2 MAIT cell TCRs were also enriched after in vitro culture, including with Mycobacterial tuberculosis. These results indicate that mature human CD4+ MAIT cells adopt distinct TCR usage from the canonical TRAV1-2+ CD8+ subset and suggest that alternative MR1 ligands in addition to riboflavin intermediates may select for them.