Multiple sclerosis is a multifactorial disorder with complex genetic basis. It is believed that genes encoding HLA molecule and cytokines are involved in the pathogenesis of MS. In this study, we have evaluated the impact of HLA-DRB1* 1501 allele and TNF-alpha - 308 G/A single nucleotide polymorphism, and their interaction, in the susceptibility to MS in Iranian population. Genomic DNA samples were prepared from whole blood of 366 MS Patients and 414 control subjects. The genotypes were determined by SSP-PCR method. Frequency of alleles and genotypes were compared between the two groups by using Fisher's exact test. HLA-DRB1* 1501 allele was more frequent among patients (OR = 1.57, P = 0.0026). TNF-α - 308 G allele and G/G genotype had higher frequency among MS patients than control subjects (G vs. A: OR = 1.26, P < 0.05); G/G vs. A/A: OR = 4.59, P = 0.0003). The odds ratio was higher among HLA-DRB1* 1501 positive individuals. Co-existence of TNF G and HLA-DRB1* 1501 alleles showed higher prevalence among MS patients (OR = 7.07, P = 0.0007). Our results have shown that HLA-DRB1*1501 allele and TNF-α - 308 G/A polymorphism are associated with the risk of multiple sclerosis in Iranian population. We also observed an interaction between these two loci that support the role of HLA alleles and cytokine genes and gene-gene interaction in the development and pathogenesis of MS.