Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors ( factor V Leiden [Arg506Gly], G20210A in the 3′-untranslated region of factor II ) and variant C677T of the methylenetetrahydrofolate reductase ( MTHFR ) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman ® assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; −/−), heterozygous (+/−) or homozygous (+/+) variants (n=362) were selected for factor V (n=115, with −/−, 40; +/−, 40; +/+, 35), factor II (n=122, with −/−, 60; +/−, 60; +/+, 2), and MTHFR (n=120, with −/−, 40; +/−, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.