Background: Anti–John Cunningham (JCV) antibodies have been detected in approximately 50% to 60% of multiple sclerosis (MS) patients. Age, sex, and geographic location have been associated with seroprevalence differences. We describe anti-JCV antibody prevalence in the Canadian cohort of patients enrolled in the JCV Epidemiology in MS study. Methods: This cross-sectional multicenter study evaluated the effects of demographic and disease characteristics on anti-JCV antibody seroprevalence in MS patients irrespective of disease type and treatment. A single blood sample was collected for analysis of anti-JCV antibodies using a two-step enzyme-linked immunosorbent assay (ELISA). Chi-square and logistic regression tests were used to determine significance. Results: A total of 4198 Canadian MS patients participated in the study; the overall anti-JCV antibody prevalence was 56.3% (95% confidence interval: 54.8% to 57.8%). Seroprevalence was significantly associated with age (increasing from 45% in young to 61% in those >60 years), sex, and region (p<0.0001 for age and sex; p=0.005 for region). No significant differences in anti-JCV antibody prevalence were associated with race, MS disease type and duration, or number and duration of treatments. Immunosuppressant use was associated with a higher seroprevalence rate (63.4%) compared with no immunosuppressant use (55.9%; p=0.040). Conclusions: Canadian MS patients had an overall anti-JCV antibody seroprevalence that was consistent with previous studies using the two-step ELISA. Significant associations of anti-JCV antibody positivity were found with age, sex, region, and immunosuppressant therapy, whereas seroprevalence was not associated with race, MS type, MS duration, or number or duration of MS treatments.