In this work, we present the use of synchotron x-ray microcomputed tomography (microCT) as a validation dataset for diffusion tensor imaging (DTI). DTI data were acquired of a post-mortem mouse brain. After metal staining, synchrotron microCT data of the sample were acquired, with 1.2 μm isotropic resolution across the whole brain. Orientation distribution functions were calculated from the microCT data using structure tensor analysis, and tractography was performed on the anterior commissure tract. Comparisons with tractography results from the diffusion MRI data show good agreement.