Oxidative stress (OS) plays an important role in Alzheimer's disease (AD) and glutathione (GSH) mitigates this effect by maintaining redox‐imbalance and free‐radical neutralization. Quantified brain GSH concentration provides distinct information about OS among age‐matched normal control (NC), mild cognitive impairment (MCI) and AD patients. We report alterations of in vivo GSH conformers, along with the choline, creatine, and N‐acetylaspartate levels in the cingulate cortex (CC) containing anterior (ACC) and posterior (PCC) regions of 64 (27 NC, 19 MCI, and 18 AD) participants using MEscher–GArwood‐Point‐RESolved spectroscopy sequence. Result indicated, tissue corrected GSH depletion in PCC among MCI (p = .001) and AD (p = .028) and in ACC among MCI (p = .194) and AD (p = .025) as compared to NC. Effects of the group, region, and group × region on GSH with age and gender as covariates were analyzed using a generalized linear model with Bonferroni correction for multiple comparisons. A significant effect of group with GSH depletion in AD and MCI was observed as compared to NC. Receiver operator characteristic (ROC) analysis of GSH level in CC differentiated between MCI and NC groups with an accuracy of 82.8% and 73.5% between AD and NC groups. Multivariate ROC analysis for the combined effect of the GSH alteration in both ACC and PCC regions provided improved diagnostic accuracy of 86.6% for NC to MCI conversion and 76.4% for NC to AD conversion. We conclude that only closed GSH conformer depletion in the ACC and PCC regions is critical and constitute a potential biomarker for AD.