Background: IDH mutations are common and prognostic in low-grade gliomas and secondary glioblastomas. IDH catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG), and subsequently 2HG. Mutant IDH preferentially catalyzes the NADPH-dependent reduction of αKG to R-2-hydroxyglutarate (R-2HG), resulting in accumulation of R-2HG, relative to its enantiomer, S-2-hydroxyglutarate (S-2HG). Previous attempts to correlate plasma/urine 2HG levels with survival and responses to therapies were limited by inconsistent results. Thus, we analyzed R- and S-2HG levels, and ratio of R- to S-2HG (RRS), in glioma tissues from patients with and without IDH mutations. Methods: Fresh frozen glioma tissues were obtained from the University of Toronto Brain Tumor Bank. Diagnosis and IDH status were determined by microscopy and immunohistochemistry. Samples were analyzed using HPLC tandem mass spectrometry coupled with a CHIROBIOTIC R column. Results: Glioma tissues from 4 patients with IDH mutations and 4 WT patients were analyzed. Patient characteristics: M:F ratio: 4:0 for mutant, 3:1 for WT. Age range at surgery: 34-59 for mutant, 47-74 for WT. Tumor size (cm): 2.3-5.4 for mutant, 2.1-8.0 for WT. Location: frontal (2), temporal (1), occipital (1) for mutant, and parietal (1), temporal (2), occipital (1) for WT. Recurrence with treatment effect: 2 of 4 mutant, 3 of 4 WT. Median survival was longer in patients with mutant IDH gliomas (39.9 vs. 17.8 months, p = 0.018). Mutant IDH gliomas had higher total 2HG (mean 451548 vs 2728 ng/g, p = 0.055) and RRS (mean 537.61 vs 0.70, p = 0.022). RRS was 0.36, 0.57, 0.70 and 1.18 in 4 WT patients, and 4.15, 522.75, 543.22 and 1080.34 in 4 mutant patients. Disease-free survival was 6.3, 6.2, 28.3 and 72.0 months respectively for mutant patients with lowest to highest RRS. Conclusions: IDH mutations result in significantly higher RRS in glioma tissues. RRS in glioma tissue, compared with plasma/urine 2HG levels, appears to be a reliable correlate of IDH status. The potential utility of RRS as a prognostic marker warrants further evaluation.