Background Medication overuse headache may be associated with widespread alterations along the thalamocortical pathway, a pathway involved in pain perception and disease progression. This study addressed whether brain metabolites in key regions of the thalamocortical pathway differed between chronic migraine patients with medication overuse headache and without medication overuse headache. Methods Magnetic resonance spectroscopic imaging was used to map metabolites in the bilateral anterior cingulate cortices, mid cingulate cortices, posterior cingulate cortices, and the thalami. Sixteen patients with medication overuse headache were compared with 16 matched patients without medication overuse headache and 16 matched healthy controls. Results Glutamate and glutamine in the right mid cingulate cortex and myo-inositol in the left anterior cingulate cortex were significantly higher in patients with medication overuse headache than patients without medication overuse headache, but similar to healthy controls. Both patient groups exhibited reduced N-acetyl-aspartate and creatine in the thalamus, reduced myo-inositol in the right anterior cingulate cortex, and elevated choline in the right mid cingulate cortex. Finally, a negative association between myo-inositol laterality index in the anterior cingulate cortices and number of days per month with acute medication use was found across all patients. Conclusions Patients with medication overuse headache were characterized by a distinct concentration profile of myo-inositol, a glial marker, in the anterior cingulate cortices that may have arisen from medication overuse and could contribute to the development of medication overuse headache.