The presence of Epstein-Barr Virus (EBV) DNA in cerebrospinal fluid (CSF) and peripheral blood (PB) samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS) and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC) positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.