Saffold cardiovirus, a newly discovered human cardiovirus, has close similarity with Theiler's murine encephalomyelitis virus (TMEV) which can cause a chronic demyelinating encephalomyelitis in mice. In this study, we tested whether Saffold cardiovirus infection of the brain is associated with multiple sclerosis (MS). Autopsy white matter samples from 19 MS and 9 normal brain donors were tested by polymerase chain reaction. All were negative. Paired cerebrospinal fluid and serum samples from 24 MS patients and 27 controls were tested for Saffold cardiovirus-specific oligoclonal bands, two patients and two controls reacted positive. We conclude that an association between Saffold cardiovirus and MS is highly improbable.