The application of multispectral imaging to discriminate myelinated and demyelinated areas of neural tissue is herein presented. The method is applied through a custom-made, multispectral imaging monochromator, coupled to a commercially available microscope. In the present work, a series of spinal cord sections were analysed derived from mice with experimental autoimmune encephalomyelitis (EAE), an experimental model widely used to study multiple sclerosis (MS). The multispectral microscope allows imaging of local areas with loss of myelin without the need of tissue labelling. Imaging with the aforementioned method and system is compared in a parallel way with conventional methods (wide-field and confocal fluorescence microscopies). The diagnostic sensitivity of our method is 90.4% relative to the 'gold standard' method of immunofluorescence microscopy. The presented method offers a new platform for the possible future development of anin vivo, real-time, non-invasive, rapid imaging diagnostic tool of spinal cord myelin loss-derived pathologies.