Clinical manifestations of antiphospholipid syndrome (APS) vary from venous and arterial thrombosis to pregnancy loss and pre-eclampsia (PEC). Our aims were to establish the prevalence of anticardiolipin antibodies (aCLA) in patients with clinical features of APS and to compare the reference ELISA method with the novel automated EliA aCLA assay. Serum samples from 1278 patients with either deep venous thrombosis (DVT), young stroke (YS), PEC, or multiple sclerosis (MS) were included. The latter cohort was included because initial presentation of APS might resemble MS. Using the internationally accepted cutoff level of 40 MPL/GPL [IgM antiphospholipid units/mL (MPL)/IgG antiphospholipid units/mL (GPL)], 0.7% and 0.5% of the samples were positive for IgM and IgG aCLA by ELISA, respectively. Using the cutoff level recommended by the manufacturer (15 MPL/GPL), the prevalence was 2.7% and 2.8%, respectively. The prevalence by EliA was 2.0% and 0.5% for IgM and IgG aCLA, respectively (40 MPL/GPL cutoff). However, with a low cutoff level, the prevalence of IgM aCLA, but not IgG aCLA, was higher in the DVT (14.3%), YS (14.6%), and PEC (17.7%) cohorts but not the MS cohort. The prevalence of aCLA was too low for reliable determination of concordance between assays. In patients with DVT, CVA, PEC, and MS, the prevalence of aCLA is low, in particular when internationally accepted cutoff values are applied. Our finding questions the efficacy of aCLA testing. Therefore, there might be either a need for defining specific clinical criteria to obtain a better rational for aCLA testing or aCLA testing should be excluded from the classification criteria.