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Acenocoumarol is widely prescribed for patients with risk of thromboembolism in Vietnam. A lot of study revealed that genetic polymorphisms of CYP2C9 and VKORC1 genes are the strongest genetic factors that have a high-impact upon the response of acenocoumarol. Therefore, in this study, we established the genotyping method of CYP2C9 and VKORC1 on Vietnamese cardiac valve replacement patients treated with acenocoumarol. Main method included DNA extraction from peripheral blood samples, polymerase chain reaction (PCR) for amplification of target genes, identifying the genotype by Sanger sequencing and restriction fragment length polymorphism PCR (PCR-RFLP). The results showed that we successfully established the optimal condition to amplify targeted polymorphism as rs1057910 of CYP2C9 gene and rs9923231, rs9934438 of VKORC1 gene.