The prothrombin G20210A mutation has been identified as a risk factor for thrombosis. We studied the relationship between prothrombin G20210A and factor V Leiden mutations in patients with thrombophilia. The first 264 patients for whom these molecular diagnostic studies were requested at our institution were included in the study. For 116 of the 264 patients, additional coagulation test results were available in the laboratory database. The prothrombin G20210A mutation was found in 16 (6.1%) of the patients and the factor V Leiden mutation in 44 (16.7%). Of the 16 patients with the prothrombin G20210A mutation, 8 also carried factor V Leiden; this association was significant. In contrast, only 2 patients of the 116 with additional coagulation testing harbored more than 1 prothrombotic risk factor. These data support the hypothesis that thrombophilia is a multigenic disorder. Among unselected samples from a Midwestern population evaluated for thrombotic risk factors, the prevalence of factor V Leiden and prothrombin G20210A mutations are similar to those found in other populations in the Western world.