Background/Aims: Researchers increasingly recognize the high frequency of comorbidity in multiple sclerosis (MS) and the negative impact on quality of life and disability, but little work has evaluated methods of comorbidity measurement in MS. We aimed to validate a self-report questionnaire for assessing comorbidity in MS. Methods: Patients with MS were recruited from the MS Clinic in Winnipeg, Canada and the Mellen Center (Cleveland Clinic, Cleveland, Ohio, USA) from October 2008 to 2009. Using a questionnaire, participants reported the presence or absence of 36 comorbidities, sociodemographic characteristics, and disability status. Abstractors blinded to questionnaire results collected data regarding the comorbidities of interest and their treatments. Using the medical record as the gold standard, we determined the sensitivity, specificity, positive and negative predictive values of the questionnaire data. To measure agreement we calculated kappa (ĸ) statistics. Results: We enrolled 404 participants. Agreement between self-report and medical records was high (ĸ >0.82) for diabetes and hypertension; substantial (ĸ = 0.62–0.80) for hyperlipidemia, thyroid disease, glaucoma, and lung disease; moderate (ĸ = 0.43–0.56) for osteoporosis, irritable bowel syndrome, migraine, depression, heart disease, and anxiety disorders. Agreement was slight to fair for the remaining comorbidities. Conclusions: Self-report is a valid way to capture comorbidities affecting MS patients.