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Glioma is the most common brain neoplasm that features aggressive behavior with a dismal prognosis. Isocitrate dehydrogenase (IDH) gene mutation in glioma is an early genetic event in gliomagenesis that occurs in virtually every tumor cell and can cause profound metabolic changes. In this manuscript, we report for the first time the analysis of Raman optical signatures of IDH genotypes for human glioma using visible resonance Raman (VRR) spectroscopy. We demonstrated that VRR is a rapid, label-free, and objective method as an alternative to the existing methods for the rapid intraoperative determination of IDH mutation status with high accuracy. This study shows AI-assisted VRR has the potential to provide a new optical molecular biomarker and perform early diagnosis of glioma, which is of great importance for current guiding surgical strategies and even for targeting in situ therapies in the future. © 2026 Wiley-VCH GmbH.