Abstract Introduction Diffusion tractography relies on complex mathematical models that provide anatomical information indirectly, and it needs to be validated. In humans, up to now, tractography has mainly been validated by qualitative comparison with data obtained from dissection. No quantitative comparison was possible because Magnetic Resonance Imaging (MRI) and dissection data are obtained in different reference spaces, and because fiber tracts are progressively destroyed by dissection. Here, we propose a novel method and software (FIBRASCAN) that allow accurate reconstruction of fiber tracts from dissection in MRI reference space. Method Five human hemispheres, obtained from four formalin-fixed brains were prepared for Klingler's dissection, placed on a holder with fiducial markers, MR scanned, and then dissected to expose the main association tracts. During dissection, we performed iterative acquisitions of the surface and texture of the specimens using a laser scanner and two digital cameras. Each texture was projected onto the corresponding surface and the resulting set of textured surfaces was coregistered thanks to the fiducial holders. The identified association tracts were then interactively segmented on each textured surface and reconstructed from the pile of surface segments. Finally, the reconstructed tracts were coregistered onto ex vivo MRI space thanks to the fiducials. Each critical step of the process was assessed to measure the precision of the method. Results We reconstructed six fiber tracts (long, anterior and posterior segments of the superior longitudinal fasciculus; Inferior fronto-occipital, Inferior longitudinal and uncinate fasciculi) from cadaveric dissection and ported them into ex vivo MRI reference space. The overall accuracy of the method was of the order of 1mm: surface-to-surface registration=0.138mm (standard deviation (SD)=0.058mm), deformation of the specimen during dissection=0.356mm (SD=0.231mm), and coregistration surface-MRI=0.6mm (SD=0.274mm). The spatial resolution of the method (distance between two consecutive surface acquisitions) was 0.345mm (SD=0.115mm). Conclusion This paper presents the robustness of a novel method, FIBRASCAN, for accurate reconstruction of fiber tracts from dissection in the ex vivo MR reference space. This is a major step toward quantitative comparison of MR tractography with dissection results.