Abstract Objective: When D-dimer is used to evaluate suspected venous thromboembolism in pregnant patients, the reference interval of common population may cause misinterpretation. The present study aims to determine reference intervals of D-dimer in the three trimesters. Methods: Four-hundred sixteen pregnant women and 32 non-pregnant women were enrolled in this cross-sectional study. Reference group had comprised 123 pregnant in the first trimester (5-11 week), 164 pregnant in the second trimester (13-20 week) and 126 pregnant women in the third trimester (25-35 week). D-dimer levels were analyzed via immunoturbidimetric assay. Results: If the threshold of 0.50 mg/L for diagnosis of VTE is used, 4.8% of pregnant women in the second trimester and 23.8% of pregnant women in the third trimester would have D-dimer levels exceeding this cut-off value. Reference intervals of D-dimer were determined as 0.11-0.40 mg/L; 0.14-0.75 mg/L and 0.16-1.3 mg/L in first, second and third trimester, respectively. Conclusion: The established D-dimer reference intervals for each trimester of pregnancy are different from those used in common population. These reference intervals may assist clinicians in making accurate clinical decisions. Further studies are needed to establish new cut-off values for the D-dimer to rule out VTE in each trimester.