To investigate structural changes in brain white matter tracts using diffusion tensor magnetic resonance imaging (DTI) in patients with overactive bladder (OAB). Treatment-seeking OAB patients and matched controls enrolled in the cross-sectional case-control Symptoms of Lower Urinary Tract Dysfunction Research Network Neuroimaging Study received a brain DTI scan. Microstructural integrity of brain white matter was assessed using fractional anisotropy (FA) and mean diffusivity (MD). OAB and urgency urinary incontinence (UUI) symptoms were assessed using the OAB Questionnaire Short-Form (OAB-q) and International Consultation on Incontinence Questionnaire-UI. The Lower Urinary Tract Symptoms (LUTS) Tool UUI questions and responses were correlated with FA values. Among 221 participants with evaluable DTI data, 146 had OAB (66 urinary urgency [UU]-only without UUI, 80 with UUI); 75 were controls. Compared with controls, participants with OAB showed decreased FA and increased MD, representing greater microstructural abnormalities of brain white matter tracts among OAB participants. These abnormalities occurred in the corpus callosum, bilateral anterior thalamic radiation and superior longitudinal fasciculus tracts, and bilateral insula and para-hippocampal region. Among participants with OAB, higher OAB-q scores were associated with decreased FA in the left inferior fronto-occipital fasciculus, P < .0001. DTI differences between OAB and controls were driven by the UU-only (OAB-dry) but not the UUI (OAB-wet) subgroup. Abnormalities in microstructural integrity in specific brain white matter tracts were more frequent in OAB patients. More severe OAB symptoms were correlated with greater degree of microstructural abnormalities in brain white matter tracts in patients with OAB.