The study assesses the relationship between thalamic proton-MR spectroscopy (H-MRS) metabolites and thalamic C-ER176 translocator-protein positron emission tomography (TSPO-PET) standardized uptake value ratios (SUVR) to advance our understanding of thalamic involvement in multiple sclerosis (MS)-associated neurodegeneration and disability. In this prospective cross-sectional study, patients with MS (pwMS) and controls underwent 3T-MRI, H-MRS, and C-ER176-PET targeting the thalamus. MRI-derived thalamic volume was normalized by intracranial volume. H-MRS metabolites-N-acetylaspartate (NAA), glutamate (Glu), glutamine (Gln), total choline (tCho), and myo-inositol (mIns)-were normalized to total creatine (tCr). Clinical disability was evaluated using MS-specific tests of Expanded Disability Status Scale-EDSS and MS-functional composite-MSFC (including Paced Auditory Serial Addition Test-PASAT). Compared to controls ( = 30), pwMS ( = 21) exhibited smaller thalamic volume, higher thalamic H-MRS mIns/tCr (putative gliosis marker), and higher thalamic C-ER176-PET SUVR (glial density marker). In pwMS, higher thalamic mIns/tCr (r = -0.67) and tCho/tCr (r = -0.52) correlated with smaller thalamic volume. In pwMS, higher thalamic mIns/tCr correlated with higher thalamic C-ER176-PET SUVR (r = 0.48) and decreased cognitive function (PASAT, rho = -0.48). In controls, decreased thalamic NAA/tCr correlated with increased thalamic C-ER176-PET SUVR (r = -0.41). Thalamus, a core central nervous system relay, is affected early in MS disease course. Glial-mediated innate immune activation in the thalamus, evaluated by increased H-MRS mIns/tCr and C-ER176-PET SUVR, is associated with loss of thalamic volume and increased disability in pwMS. The multimodal imaging approach with H-MRS mIns/tCr and C-ER176-PET SUVR emerges as potential glial biomarkers, to better understand disease mechanisms and evaluate therapeutic interventions targeting glial activity in MS.