Objective: A study was undertaken to define the prevalence of anti‐JC virus (JCV) antibodies in multiple sclerosis (MS) patients and to evaluate the analytical false‐negative rate of a 2‐step anti‐JC virus antibody assay. Methods: STRATIFY‐1 is an ongoing, longitudinal, observational study of relapsing MS patients in the United States who are being treated or considering treatment with natalizumab. Baseline serum and plasma samples were collected for anti‐JC virus antibody detection using an analytically validated, 2‐step, virus‐like particle‐based enzyme‐linked immunosorbent assay. Urine was collected for JC virus DNA detection. Results: At baseline (n = 1,096), overall anti‐JC virus antibody prevalence was 56.0% (95% confidence interval CI, 53.0‐59.0) in STRATIFY‐1 patients, with an assay false‐negative rate of 2.7% (95% CI, 0.9–6.2). Prevalence was significantly lower in females (53.4%; 95% CI, 49.9–56.8) than males (64.3%; 95% CI, 58.2–70.0) and increased with age, p = 0.0019 and p = 0.0001, respectively. Prevalence was similar in patients regardless of natalizumab exposure or prior immunosuppressant use, p = 0.9709 and p = 0.6632, respectively. STRATIFY‐1 results were generally consistent with those observed in another large North American cohort, TYGRIS‐US (n = 1,480). Interpretation: Baseline results from STRATIFY‐1 are consistent with other studies utilizing this assay that demonstrate a 50 to 60% prevalence of anti‐JC virus antibodies, a low false‐negative rate, and an association of increasing age and male gender with increasing anti‐JC virus antibody prevalence. Neither natalizumab exposure nor prior immunosuppressant use appear to affect prevalence. Longitudinal data from STRATIFY‐1 will confirm the stability of anti‐JC virus antibody prevalence over time. ANN NEUROL 2011