D-dimer, the final degradation product of cross-linked fibrin, is typically elevated in patients with acute venous thromboembolism. With its high sensitivity and negative predictive value, D-dimer testing may have a role for ruling-out the diagnosis in patients with suspected deep vein thrombosis or pulmonary embolism. For this purpose, D-dimer testing has been integrated in sequential diagnostic strategies including those using pretest clinical probability assessment and imaging techniques. A large variety of assays are now available for D-dimer measurement, with different sensitivities and specificities for the diagnosis of venous thromboembolism. Attempts to standardize the various D-dimer assays have been made but without any definitive answers as yet. The diagnostic yield of D-dimer testing is affected not only by the choice of the appropriate assay but also by patient characteristics. As a consequence, the clinical usefulness of D-dimer testing for the exclusion of suspected venous thromboembolism should be carefully evaluated in special clinical settings.