A multi-site study to assess the accuracy and performance of the biplex Invader assay for genotyping five polymorphisms implicated in venous thrombosis was carried out in seven laboratories. Genotyping results obtained using the Invader biplex assay were compared to those obtained from a reference method, either allele-specific polymerase chain reaction (AS-PCR), restriction fragment length polymorphism (PCR-RFLP) or PCR-mass spectrometry. Results were compared for five loci associated with venous thrombosis: Factor V Leiden, Factor II (prothrombin) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, and plasminogen activator inhibitor (PAI-1) 4G/5G. Of a total of 1448 genotypes tested in this study, there were 22 samples that gave different results between the Invader biplex assay and the PCR-based methods. On further testing, 21 were determined to be correctly genotyped by the Invader Assay and only a single discrepancy was resolved in favor of the PCR-based assays. The compiled results demonstrate that the Invader biplex assay provides results more than 99.9% concordant with standard PCR-based techniques and is a rapid and highly accurate alternative to target amplification-based methods.