Multiple Sclerosis (MS) is characterized by demyelination and neurodegeneration of the central nervous system and causes excessive fatigue in more than 80% of the patients. The pathophysiologic mechanisms causing fatigue are still largely unknown. In 46 right-handed patients with relapsing-remitting MS and 25 right-handed controls, we performed diffusion MRI and applied streamline based probabilistic tractography to derive unilateral anatomical connectivity maps for the white matter of the right and left hemispheres. The maps provide an indication how often a streamline has passed through a given voxel. Since tractography based anatomical connectivity mapping (ACM) is sensitive to disease-induced changes in anatomical connectivity, we used ACM to test whether motor fatigue is associated with altered ipsi-hemispherical anatomical connectivity in the major motor output pathway, the corticospinal tract (CST). Patients had higher mean ACM values in the CST than healthy controls. This indicated that a higher number of streamlines, starting from voxels in the same hemisphere, travelled through the CST and may reflect an accumulated disease-induced disintegration of CST. The motor subscale of the Fatigue Scale for Motor and Cognitive functions (FSMC_MOTOR) was used to define sub-groups with (n = 29, FSMC_MOTOR score ≥ 27) and without motor fatigue (n = 17, FSMS_MOTOR score ≤ 26). Patients without fatigue only showed higher ACM values in right CST, while mean ACM values were unaltered in left CST. The higher the mean ACM values in the left relative to the right CST, the more patients reported motor fatigue. Left-right asymmetry in anatomical connectivity outside the CST did not scale with individual motor fatigue. Our results link lateralized changes of tractography-based microstructural properties in the CST with motor fatigue in relapsing-remitting MS.