Methylenetetrahydrofolate reductase (MTHFR) and tumor necrosis factor-alpha (TNF-α) play major roles in cardiovascular and inflammatory disorders. This study aimed to develop a new multiplex PCR genotyping method for the simultaneous detection of MTHFR 677C>T and TNF-α -308G>A, which are the two single nucleotide polymorphisms (SNPs) that are widely known to confer susceptibility to major vascular and inflammatory disorders. DNA was amplified using multiplex PCR, which was optimized by evaluations of the annealing temperature, the effects of various magnesium chloride, primer and enzyme concentrations, and the amount of DNA template. Restriction fragment length polymorphism (RFLP) analysis was performed in two separate tubes followed by agarose gel electrophoresis. One hundred twenty-nine healthy volunteers were recruited, and the MTHFR 677C>T and TNF-α -308G>A variants were genotyped using a novel multiplex PCR-RFLP technique. The results were confirmed by DNA sequencing. The allele frequencies of MTHFR 677C>T were 97.29% (C allele) and 2.71% (T allele). For TNF-α -308G>A, the allele frequencies were 98.45% (G allele) and 1.55% (A allele). The PCR-RFLP method developed in this study is simple, cost-effective and time-saving. It can be used to simultaneously genotype subjects for the MTHFR 677C>T and TNF-α -308G>A variants with 100% concordance with DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677C>T and TNF-α -308G>A variants.