Purpose To evaluate the ability of high-gradient-diffusion magnetic resonance (MR) imaging by using gradient strengths of up to 300 mT/m to depict axonal disease in lesions and normal-appearing white matter (NAWM) in patients with multiple sclerosis (MS) and to compare high-gradient-diffusion MR findings in these patients with those in healthy control subjects. Materials and Methods In this HIPAA-compliant institutional review board-approved prospective study in which all subjects provided written informed consent, six patients with relapsing-remitting MS and six healthy control subjects underwent diffusion-weighted imaging with a range of diffusion weightings performed with a 3-T human MR imager by using gradient strengths of up to 300 mT/m. A model of intra-axonal, extra-axonal, and free water diffusion was fitted to obtain estimates of axon diameter and density. Differences in axon diameter and density between lesions and NAWM in patients with MS were assessed by using the nonparametric Wilcoxon matched-pairs signed rank test, and differences between NAWM in subjects with MS and white matter in healthy control subjects were assessed by using the Mann-Whitney U test. Results MS lesions showed increased mean axon diameter (10.3 vs 7.9 μm in the genu, 10.4 vs 9.3 μm in the body, and 10.6 vs 8.2 μm in the splenium; P < .05) and decreased axon density ([0.48 vs 1.1] × 10(10)/m(2) in the genu, [0.40 vs 0.70] × 10(10)/m(2) in the body, and [0.35 vs 1.1] × 10(10)/m(2) in the splenium; P < .05) compared with adjacent NAWM. No significant difference in mean axon diameter or axon density was detected between NAWM in subjects with MS and white matter in healthy control subjects. Conclusion High-gradient-diffusion MR imaging using gradient strengths of up to 300 mT/m can be used to characterize axonal disease in patients with MS, with results that agree with known trends from neuropathologic data showing increased axon diameter and decreased axon density in MS lesions when compared with NAWM. (©) RSNA, 2016 Online supplemental material is available for this article.