The risk of progressive multifocal leukoencephalopathy (PML) in patients treated with natalizumab for multiple sclerosis (MS) is a serious concern. The presence of anti-JC virus antibodies is a risk factor for PML development, but 2.5 % of the patients result falsely-negative, while the prognostic relevance of testing JCV-DNA in biological fluids of treated patients is debated. Aim of this work was to evaluate the utility of testing JCV-DNA, together with anti-JCV antibodies, in biological samples of treated patients as a tool for PML risk stratification. 126 subjects from 5 MS Centers in Italy were included in the study. We performed a cross-sectional study in 63 patients testing JCV-DNA in blood, peripheral blood cells and urine. We longitudinally assessed the presence of JCV-DNA in a cohort of 33 subjects, one of which developed PML. We could test retrospectively serum samples from another PML case occurred during natalizumab therapy. Anti-JCV antibodies and urinary JCV-DNA were both tested in 73 patients. No changes in JCV-DNA status occurred during natalizumab treatment. The subject who developed PML in the longitudinal cohort had detectable JCV-DNA in urine at all time-points while serum or blood from both PML patients were always negative before the onset of disease and, in one case, after. Four subjects with JCV-DNA in urine and undetectable anti-JCV antibodies were retested for anti-JCV antibodies and three out of four resulted positive. In conclusion, testing JCV-DNA in urine is complementary to testing anti-JCV antibodies in identifying patients at risk of PML.