Aim: In this study, we aimed to reveal the neuroimaging and cerebrospinal fluid (CSF) characteristics in the differential diagnosis of multiple sclerosis (MS) and migraine. Material and Methods: We retrospectively analyzed prospectively collected data from our referral center for individuals with an initial diagnosis of demyelinating disease and eventually not diagnosed with MS and who were diagnosed with migraine without aura according to the International Classification of Headache Disorders, 3rd edition criteria. As a control group, consecutive individuals registered in our iMed database who were diagnosed with MS according to the 2017 McDonald criteria without migraine were enrolled. Results: Fifty-five individuals with migraine-associated white matter lesions (MAWML) and 55 patients with (pw) MS were included in the study as a control group. The mean age and gender distributions were similar between groups (p=0.212 and 0.864, respectively). Oligoclonal bands (OCBs) were detected in 29 (52.7%) pwMS. In eight (14.5%) pwMS, CSF was normal for OCBs (p<0.0001). Paraventricular lesions, capping lesions, lesions smaller than 3 mm, and subcortical lesions were significantly more frequent in the MSWML group (p=0.001, 0.001, 0.001, and 0,01, respectively). Cortical/juxtacortical lesions, periventricular lesions, middle cerebellar peduncle lesions, infratentorial lesions, and callosal lesions were more frequent in pwMS (p=0.004, 0.0001, 0.001, 0.001, and 0.013, respectively). Discussion: In this study, the contribution of CSF findings and magnetic resonance imaging (MRI) lesion locations has been shown in the differential diagnosis of MS and migraine as a guide for MS-specific neurologists and neuroradiologists.