<h3>Objective:</h3> In this study we investigated ambulation and its association with white matter tracts in eighty Relapsing Remitting Multiple Sclerosis (RRMS) patients. <h3>Background:</h3> Ambulation is commonly affected in Multiple Sclerosis (MS) and is a major determinant of disability. Timed 25-foot walk (T25FW) is an objective measure of maximum walking speed in a short distance. To date, consistent neuroimaging correlates of ambulation in MS are not well explored. <h3>Design/Methods:</h3> Eighty participants with RRMS were recruited and underwent a 3 Tesla whole brain MRI scan, Expanded Disability Status Scale (EDSS), and T25FW. We performed region-based tractography and we correlated Mean Fractional Anisotropy (FA) and Mean Diffusivity (MD) to the patients’ T25FW, using the Spearman’s rank correlation coefficient (r). <h3>Results:</h3> Our participants’ mean age was 42±10.5 years, median EDSS was 1.4 (1–4), and disease duration was 8.4±8 years. The strongest correlations between FA and T25FW were observed on the left arcuate fasciculus (r=−0.5, p= 0.001), right and left acoustic radiation (r=−0.41, p=0.001; r=−0.38, p=0.001 respectively), right and left corticothalamic pathway (r=−0.41, p=0.001; r=−0.37, p=0.001 respectively), right optic radiation (r=−0.41, p=0.001) and medial lemniscus (r=−0.35, p=0.001). The strongest correlations between MD and T25FW were found on the right and left arcuate fasciculus (r=0.40, p= 0.001 and r=0.45, p= 0.001 respectively), right and left corticothalamic pathway (r=0.37, p=0.001; r=0.35, p=0.001 respectively), right dorsal longitudinal fasciculus (r=0.36, p=0.001; r=0.35), and superior cerebellar peduncle (r=0.40, p=0.001). <h3>Conclusions:</h3> Our findings enrich our understanding of ambulation in MS and confirm that ambulation is a complex process. Longitudinal studies are needed to validate our findings and further determine the role of different white matter tracts in its pathobiology. <b>Disclosure:</b> Miss Jageka has nothing to disclose. Fen Bao has nothing to disclose. Mr. Elkhooly has nothing to disclose. Dr. Raghib has nothing to disclose. Ms. Pelc has nothing to disclose. Mr. Lazar has nothing to disclose. Zahid Latif has nothing to disclose. Ms. Santiago-Martinez has nothing to disclose. Dr. Rube has nothing to disclose. Dr. Bernitsas has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Janssen@Janssen. Dr. Bernitsas has received personal compensation in the range of $5,000-$9,999 for serving on a Speakers Bureau for Biogen. The institution of Dr. Bernitsas has received research support from Roche/Genentech.