Gray matter (GM) damage is an important pathophysiological feature in Multiple Sclerosis (MS), and may be related to clinical, including cognitive, deficits. Quantitative single-voxel 1H-Magnetic Resonance Spectroscopy (TR/TE 6000/20 ms) was performed in 33 MS patients (11 per disease subtype; mean age 48 years, 16 females) and 10 healthy controls (mean age 43 years, 7 females). No overall spectroscopic changes were found in MS cortex. In MS thalamus, a 9% decrease of N-acetyl aspartate (NAA; P = 0.005) and a 31% increase of myo-inositol (Ins; P = 0.002) were found. A 21% Ins increase was observed (P = 0.02) in MS hippocampus. Reduced NAA and increased Ins concentrations are thought to reflect neuro-axonal damage or loss and gliosis, respectively. Significant correlations between Ins concentrations and total-brain T2 lesion load were found for MS thalamus (r = 0.65, P < 0.001) and hippocampus (r = 0.57, P = 0.001). MS thalamic and hippocampal Ins concentrations also correlated with each other (r = 0.68; P < 0.001). Cortical GIn correlated with thalamic NAA (r = -0.38; P = 0.03) in MS. Thalamic and hippocampal Ins increases were most prominent in secondary-progressive (SP) patients (37% and 34%, respectively), whereas the largest thalamic NAA decrease (14%) was found in primary-progressive (PP) patients. In conclusion, thalamic and hippocampal GM pathology are important features of (progressive) MS.