Introduction: Gene polymorphisms in human methylenetetrahydrofolate reductase (MTHFR) have been linked to the disorders of folate metabolism, and may contribute to such disease as neural tube defect, coronary heart disease, venous thrombosis, and several types of cancers. Methodology: We have modified our current real-time PCR method (Clin Biochem 2000;33:535-539.) for the detection of the MTHFR c.677C > T variant to a real-time multiplex-PCR method for detection of MTHFR c.1298A > C and c.677C > T variants on a LightCycler 1.2. Our multiplex method is partly based on that of Agarwal et al.(J Mol Diag 2007;9:345). Results: The genotyping results of c.677C > T variant analyzed by both methods were in agreement. All samples analyzed for the c.1298A > C variant by the Multiplex-PCR method gave clear-cut results. The LightCycler multiplex method was validated with 14 specimens previously genotyped by restriction fragment length polymorphism at Montreal Children’s Hospital. All results were in agreement except for one sample that failed to amplify. There was no significant difference in Tm between the Multiplex-PCR procedure and the previously reported Tm’s for both variants. When run alone but not as a multiplex, the progress curve of the c.1298A > C variant exhibited the “hook effect”. Asymmetric amplification was found to reduce this effect. Conclusions: The multiplex assay is reliable, economical, fast and simple method to perform as compared with RFLP technique. Asymmetric PCR is a helpful tool to minimize the hook effect.