Interferons-β (IFN-β) are the most widely used immunomodulatory drugs for treatment of multiple sclerosis (MS). The development of neutralizing antibodies (NABs) against IFN-β is one of the main reasons for treatment failure. While formulation of the drug has a proven impact on the development of NABs, the genetic predisposition to develop antibodies is poorly understood. We performed genome-wide single-nucleotide polymorphism (SNP) genotyping in 362 MS patients of whom 178 had developed and 184 had not developed antibodies on IFN-β therapy. Four candidate SNPs were validated in an independent cohort of 350 antibody-positive and 468 antibody-negative MS patients. One SNP within the human leucocyte antigen (HLA) region (rs9272105, P-value: 3.56 × 10⁻¹⁰) and one SNP in an intergenic region on chromosome 8q24.3 (rs4961252, P-value: 2.92 × 10⁻⁸ showed a genome-wide significant association with the anti-IFN-β antibody titers. We found no interaction between the genome-wide significant SNPs (rs9272105 and rs4961252) in our study and the previously described HLA-DR*0401 or *0408 alleles, indicating an additive effect of SNPs and HLA alleles. Testing for these SNPs and the HLA-DR*0401 or *0408 alleles allows to identify patients at risk to develop antibodies to IFN-β and may provide helpful information for individual treatment decisions.