Methylene Tetra Hydro Folate Reductase (MTHFR) is an enzyme that in humans is encoded by the MTHFR gene. MTHFR catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. Genetic variation in this gene influences susceptibility to occlusive vascular diseases, neural tube defects, colon cancer and acute leukemia, and mutations. This gene is associated with Methylenetetrahydrofolate reductase deficiency. Two kind of functional polymorphism, 677 C > T and 482 G > T have shown effect to severe disease in Human including cancers. Because of this enzyme deficiency cause many problems, till now 40 types of fatal disorders have been found in Human species. Main aim to do this project is to develop the new and novel method of detection of MTHFR gene related mutations at early stage. For this, 25 peripheral blood samples were randomly collected among the Indian population and DNA was isolated and the genotype polymorphism was determined by Multiplex-PCR RFLP method.