Signal abnormalities on magnetic resonance imaging (MRI) T2-weighted images (T2WI) have been described in patients with Creutzfeldt-Jakob disease; however, the pathology underlying these findings remains to be fully described. We investigated the time-course of signal alterations in a murine model of prion disease using in vivo 9.4 Tesla micro magnetic resonance imaging (muMRI). The topography of muMRI signal changes was correlated with the accumulation of proteinase resistant PrP(Sc) in corresponding brain sections. Increased signal intensity on T2WI was observed in the septum and in the hippocampus of presymptomatic mice 120 days post infection (dpi). Mildly symptomatic animals (150 dpi) and animals with apparent neurological deficit (180 dpi) had a greater increase of signal intensity on T2WI in the septum and the hippocampus; in addition, abnormalities in the cortex and in the thalamus were found. Neuropathological evaluation demonstrated accumulation of PrP(Sc) and astrogliosis but only minimal or no spongiform changes in structures where abnormal signal was detected. These observations suggest that early pathological changes related to the accumulation of PrP(Sc) may be detectable in presymptomatic subjects using MRI systems with higher magnetic field strength.