A number of groups have examined the pathological substrate of signal changes on magnetic resonance imaging (MRI) in post-mortem (PM) brain of patients with multiple sclerosis (MS). Such studies will benefit from using a standardized method to reliably co-register regions of interest on MRI and tissue specimens. We investigated the usefulness of a stereotactic navigation system for this purpose. We also addressed the sensitivity of different standard MRI sequences with regard to lesion conspicuity in PM MS brain. Post-mortem brains of eight patients with MS were studied. Formalin-fixed coronal slices were placed in the head frame of a stereotactic system. Proton density-, T2-weighted and fast fluid-attenuated inversion recovery (FLAIR) scans were obtained and visually matched with scans that had been previously obtained on the same, but fresh, specimens. Guided by the stereotactic target points, the dissection of the fixed specimens was performed. After processing the blocks for embedding in paraffin, sections were stained with haematoxylin-eosin and Luxol fast blue. T2-weighted MRI of fixed brain revealed 24 areas suspected to be MS lesions, all of which were confirmed histologically. Three of these lesions were not visible on macroscopic inspection. There were 14 additional hyperintensities on T2-weighted or FLAIR MRI of the fresh specimens, five of which did not correlate to MS lesions histologically. Stereotactic navigation is a useful approach to co-register MRI and histopathology in PM brain of MS patients and may improve the precision of MRI-guided sampling of tissue specimens. Standard T2-weighted MRI appeared to be the single most useful approach for lesion detection in fresh and fixed specimens.