Study objectiveTo estimate the diagnostic accuracy of three-dimensional ultrasonography (3D US) compared to hysteroscopy/laparoscopy, in the investigation of uterine congenital anomalies using the ESHRE/ESGE classification of female genital tract congenital anomalies. DesignProspective blind, comparative, cohort study. SettingUniversity Tertiary Hospital and affiliated private Hospital.Patients and methodsSixty-two women consecutively referred with a suspected diagnosis of uterine congenital anomalies. The ESHRE/ESGE classification of congenital anomalies of the female genital tract was used for the description of abnormal findings.InterventionsAll patients underwent (1) 3D US and (2) hysteroscopy with laparoscopy to establish the final diagnosis.ResultsConcordance between 3D US and hysteroscopy with laparoscopy about the type and the classification of uterine anomaly was verified in 61 cases, including all those with septate uterus, dysmorphic uterus, bicorporeal, hemi-uterus or unicorporeal, and aplastic uterus and one out of two with normal uterus. For the diagnosis of septate uteri, which was the most common anomaly, the sensitivity of 3D US was 100%, the specificity was 92.3%, the PPV was 98% and the NPV was 100%, with kappa index 0.950. For bicorporeal, dysmorphic uterus, hemi-uteri or unicorporeal and aplastic uterus the sensitivity, specificity, PPV and NPV were all 100% with K=1.00. Overall, 3D US showed perfect diagnostic accuracy (Kappa index=0.945) in the detection of congenital uterine anomalies.Conclusion3D US appears to be a very accurate method for the diagnosis of congenital uterine anomalies.