Our aim in the present study is to evaluate the variation present in the TLR4 gene and its relationship with multiple sclerosis susceptibility in the Spanish population. Three hundred and sixty-two multiple sclerosis patients and 467 healthy controls from the Spanish population were included in the present study. Twelve single nucleotide polymorphisms (SNPs) were selected, and analyzed by the TaqMan technique. No statistically significant differences for any polymorphism or haplotypes were observed when patients were compared with controls. In conclusion, TLR4 does not play a major role in the predisposition to suffer from multiple sclerosis in our population.