Although significant improvements have been made regarding the visualization and characterization of cortical multiple sclerosis (MS) lesions using magnetic resonance imaging (MRI), cortical lesions (CL) continue to be under-detected in vivo, and we have a limited understanding of the causes of GM pathology. The objective of this study was to characterize the MRI signature of CLs to help interpret the changes seen in vivo and elucidate the factors limiting their visualization. A quantitative 3D high-resolution (350 μm isotropic) MRI study at 3 Tesla of a fixed post mortem cerebral hemisphere from a patient with MS is presented in combination with matched immunohistochemistry. Type III subpial lesions are characterized by an increase in T1, T2 and M0, and a decrease in MTR in comparison to the normal appearing cortex (NAC). All quantitative MR parameters were associated with cortical GM myelin content, while T1 showed the strongest correlation. The histogram analysis showed extensive overlap between CL and NAC for all MR parameters and myelin content. This is due to the poor contrast in myelin content between CL and NAC in comparison to the variability in myelo-architecture throughout the healthy cortex. This latter comparison is highlighted by the representation of T1 times on cortical surfaces at several laminar depths.