In response to microbial infection, the non-classical antigen presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal associated invariant T (MAIT) cells. Here, we further explore the repertoire of ligands captured by MR1 produced from Mycobacterium smegmatis via mass spectrometry. We identify the novel MR1 ligand “Photolumazine V” (PLV), a hydroxyindolyl-ribityllumazine with four possible isomers differing in the positioning of a hydroxyl group, all of which can be produced by M. smegmatis. Synthetic PLV isomers induce MR1 surface translocation and MAIT cell clones expressing distinct TCR beta chains differentially responded to the PLV isomers. These data demonstrate that subtle changes in the positioning of a single hydroxyl group modulate TCR recognition and emphasize how structural microheterogeneity in the MR1 antigen repertoire influences MAIT cell recognition.