In multiple sclerosis (MS) more than 95% of the patients have positive oligoclonal bands (OCB) in the cerebrospinal fluid (CSF). Previous studies have reported differences between patients with and without OCB mainly with regard to clinical parameters such as age, gender, disease duration, and clinical severity. However, several MRI characteristics have also been hypothesized to be distinct, and a varying lesion load in OCB-negative and -positive patients is proposed. In this study, we aimed to evaluate whether Barkhof's diagnostic MRI criteria are unequally frequently fulfilled in OCB-negative and -positive MS patients. We screened our database for all OCB-negative MS patients who had (1) been treated with the diagnosis of a clinical definite relapsing-remitting MS in our institution as well as (2) undergone CSF analysis and MR brain imaging during hospital stay between January 2004 and December 2007. Eleven OCB-negative patients were identified who fulfilled these criteria. In a second step, we carefully matched each of them to two OCB-positive controls according to age, gender, EDSS, and disease duration. The separate analysis of the several parameters of Barkhof's criteria revealed a less frequent prevalence of infratentorial (3/11 vs. 18/22; P = 0.005) and a more frequent occurrence of juxtacortical lesions (10/11 vs. 10/22; P = 0.022) in OCB-negative as compared to OCB-positive patients. The overall fulfillment of the Barkhof criteria did not differ in OCB-negative and -positive patients (7/11 vs. 16/22; P = 0.696). Further analyses of MRI findings between OCB-negative and -positive MS patients might contribute to a better pathophysiological understanding of the genesis and evidence of OCB in the CSF of MS patients.