Lower urinary tract (LUT) symptoms are reported in more than 80% of patients with multiple sclerosis (MS), most commonly an overactive bladder (OAB). The relationship between brain white matter (WM) changes in MS and OAB symptoms is poorly understood. We aim to evaluate (i) microstructural WM differences across MS patients (pwMS) with OAB symptoms, patients without LUT symptoms, and healthy subjects using diffusion tensor imaging (DTI), and (ii) associations between clinical OAB symptom scores and DTI indices. Twenty-nine female pwMS [mean age (SD) 43.3 years (9.4)], including seventeen with OAB [mean age (SD) 46.1 years (8.6)] and nine without LUT symptoms [mean age (SD) 37.5 years (8.9)], and fourteen healthy controls (HCs) [mean age (SD) 48.5 years (20)] were scanned in a 3T MRI with a DTI protocol. Additionally, clinical scans were performed for WM lesion segmentation. Group differences in fractional anisotropy (FA) were evaluated using tract-based spatial statistics. The Urinary Symptom Profile questionnaire assessed OAB severity. A statistically significant reduction in FA (p = 0.004) was identified in microstructural WM in pwMS, compared with HCs. An inverse correlation was found between FA in frontal and parietal WM lobes and OAB scores (p = 0.021) in pwMS. Areas of lower FA, although this did not reach statistical significance, were found in both frontal lobes and the rest of the non-dominant hemisphere in pwMS with OAB compared with pwMS without LUT symptoms (p = 0.072). This study identified that lesions affecting different WM tracts in MS can result in OAB symptoms and demonstrated the role of the WM in the neural control of LUT functions. By using DTI, the association between OAB symptom severity and WM changes were identified, adding knowledge to the current LUT working model. As MS is predominantly a WM disease, these findings suggest that regional WM involvement, including of the anterior corona radiata, anterior thalamic radiation, superior longitudinal fasciculus, and superior frontal-occipital fasciculus and a non-dominant prevalence in WM, can result in OAB symptoms. OAB symptoms in MS correlate with anisotropy changes in different white matter tracts as demonstrated by DTI. Structural impairment in WM tracts plays an important role in LUT symptoms in MS.