Phosphomethylpyrimidine synthase (ThiC) is a noncanonical radical SAM enzyme that catalyzes the complex radical rearrangement of aminoimidazole ribonucleotide (AIR) to hydroxymethylpyrimidine phosphate (HMP-P) as part of the thiamine biosynthetic pathway in bacteria. In this work, we investigate the mechanism of ThiC using advanced electron paramagentic resonance (EPR) techniques. Freeze-quenching a reaction of ThiC revealed the accumulation of a new radical species. By employing electron nuclear double resonance (ENDOR) spectroscopy with various substrate isotopologues, we determined the hyperfine parameters of several nuclei that allowed us to propose a structure for this intermediate. The accumulated species was characterized as an aminoimidazole centered radical that was sensitive to perturbations in the H-bonding network. In addition, mutagenesis of active site residues resulted in the accumulation of two distinct early intermediates, including a C5′ ribonucleotide centered radical and a ribose C2′ radical fragment. Our results highlight the importance of outer coordination sphere interactions for stabilizing substrate radical intermediates, which is critical to the function of ThiC. Identification of these early radical intermediates provides insights into the initial steps of the ThiC mechanism.