A laboratory investigation of thrombophilia comprises hereditary and acquired laboratory markers. The first category includes the factor V Leiden mutation, prothrombin mutation, hereditary antithrombin deficiency, protein C deficiency, protein S deficiency and certain congenital dysfibrinogenemias; however, hereditary thrombophilias are of little value as indicators of an elevated risk of recurrent thrombosis. The second category of acquired thrombophilia includes antiphospholipid antibody syndrome, myeloproliferative neoplasms and paroxysmal nocturnal hemoglobinuria. The correct indications and timing of the investigation as well as consideration of the clinical circumstances and preanalytical influencing factors are of major importance for a valid interpretation of laboratory results.