The frequency and the consequences of binding and neutralizing antibodies (BAbs and NAbs) against Interferon beta (IFNbeta) in Turkish multiple sclerosis (MS) patients have not been determined yet, which could differ in such a country which is between Europa and Asia. The aim of the study is to assess the frequency of these antibodies, and to evaluate the impact of NAbs, from the clinical and radiologic aspects in Turkish patients with MS. One hundred and two MS patients were included. BAbs were screened using capture enzyme-linked immunosorbent assay (cELISA), and NAbs were detected via Myxovirus protein A (MxA) messenger RNA (mRNA) induction assay (real-time polymerase chain reaction-PCR) at the beginning and one year later. Relapse rate and expanded disability status scale (EDSS) were used to assess the clinical impact. Gadolinium enhanced lesions and T2 lesion volume were used as magnetic resonance imaging (MRI) parameters. Persistent NAb positivity defines to be positive both at first and then one year later. NAbs were detected in 12.2% (6/49) of IFNbeta-1b treated patients, and in 7.5% (3/40) of IFNbeta-1a SC treated patients, but none of the IFNbeta-1a IM treated patients had detectable NAbs. It was found that the mean relapse rate difference was significantly higher in persistent NAb negative patients (p = 0.024). Persistent NAb positivity had no effect on T2 lesion volume and contrast enhancing lesions. 60% of the persistent NAb positive patients had at least one relapse during one-year of follow-up. On the other hand, 32% of persistent NAb negative patients were detected to have at least one relapse. Data from this study suggest that patients may become unresponsive to IFNbeta therapy even when the frequency of NAbs does not prove to be as high as those in the literature. Nevertheless, one should keep in mind that disease activity is not always equal to NAb positivity.