We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia.