A point mutation, 1691 G --> A in the coagulation factor V gene results in an Arg506 --> Gln amino acid substitution in the factor V molecule. This mutation, defined as factor VLEIDEN, results in activated protein C (APC) resistance and is the most common genetic risk factor for familial thrombophilia. A new mini-sequencing method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed for the screening of the 1691G --> A substitution in factor V. In this method, a fragment of genomic DNA containing the 1691st base is first amplified, followed by mini-sequencing in the presence of dGTP and ddATP, ddCTP, and ddTTP. In this manner, the primer is extended by one base from one allele and two bases from the other allele. The extended products are analyzed using MALDI-TOF mass spectrometry. The base at position 1691 is identified based on the number of nucleotides added. We have used this method to genotype 16 APC-resistant patients previously identified by conventional methods and 11 normal control samples. The genotypes of all samples were correctly identified. This method is accurate, fast, and potentially allows for simultaneous multiplex genotyping of a number of mutation sites associated with thrombophilia and clot formation.