<p>Molybdenum isotopes, particularly ⁹⁶Mo, ⁹⁷Mo, and ⁹⁸Mo—the precursors for the medical isotope ^99mTc—are vital in healthcare, science of materials, and nuclear research. While electromagnetic separation efficiently enriches these isotopes, analyzing their abundance requires techniques sensitive to subtle changes in electronic structure caused by isotopic mass variations. In this study, X-band EPR spectroscopy was experimentally employed to provide a suitable and sensitive method for probing these effects. We enriched ⁹⁶Mo, ⁹⁷Mo, and ⁹⁸Mo isotopes electromagnetically and purified the collected powders through a chemical refining process. The refined samples were characterized using XRD, SEM, ICP, ICP-MS, PL, and EPR. Ab initio calculations were applied to investigate the electronic, optical, and EPR properties with the computed band gaps, optical characteristics, and EPR spectra validated against experimental results. Our calculations identified specific native defects in the α-MoO₃ crystal lattice. Crucially, only X-band EPR measurements were experimentally performed and the high-frequency (W- and J-band) EPR spectra were generated through simulations by using DFT/MD-derived EPR parameters, which allowed an assessment of the potential spectroscopic signatures of different Mo isotopes in α-MoO₃ and suggest that high-frequency EPR could, in principle, serve as a tool for isotopic analysis.</p>