Objective: To find the strongest neuroimaging predictors for motor dysfunction using conventional and quantitative imaging measures focusing on the corticospinal tract (CST) in a large cohort of patients with long-standing multiple sclerosis (MS). Methods: In this cross-sectional study, a wide spectrum of neuroimaging measures at the whole-brain, cervical, and CST level were analyzed in 195 patients with MS and 54 healthy controls. Motor function was assessed using the Expanded Disability Status Scale (EDSS), 9-Hole Peg Test, Timed 25-Foot Walk Test, and Multiple Sclerosis Walking Scale. Associations between damage in different parts of the motor system and motor functioning were assessed using stepwise linear regression. Results: Patients had an average disease duration of 19.98 (±6.99) years and a median EDSS score of 4 (range: 1.0-8.0). EDSS score was associated with number of infratentorial and cervical cord lesions, lesion volume in the CST, and mean upper cervical cord area (adjusted R 2 0.403). Timed 25-Foot Walk Test score was associated with number of infratentorial lesions and cerebellar volume (adjusted R 2 0.150), 9-Hole Peg Test score with number of infratentorial lesions and thickness of the cortex connected to the CST (adjusted R 2 0.245), and Multiple Sclerosis Walking Scale with number of infratentorial and cervical lesions, thickness of the cortex connected to the CST, and mean upper cervical cord area (adjusted R 2 0.354). Conclusions: Motor dysfunction in MS has a complex substrate that cannot be ascribed to a single neuroimaging finding, but is the consequence of infratentorial and spinal cord damage, as well as damage in the CST.