Objective: To establish reference values for the volumes of fetal cardiac ventricular walls using three-dimensional ultrasonography (3DUS) and perform data validation using fetuses with confirmed congenital heart disease (CHD). Methods: This prospective, cross-sectional study analyzed 371 singleton pregnancies between 20w0d and 33w6d of gestation. Ventricular volumes were assessed using spatiotemporal image correlation (STIC) and virtual organ computer-aided analysis (VOCAL). We calculated the polynomial regressions, adjusted by the coefficient of determination (R2). To assess intra-observer concordance, the intraclass correlation coefficient (ICC) was used. To validate the curves, the ventricular wall volumes of 22 fetuses with CHD were evaluated. Results: There was a good correlation between these mean volumes and gestational age, and this correlation was best represented by linear equations. Intra-observer concordance in assessing the volumes of the right (ICC=0.90) and left ventricular walls (ICC=0.97) was good. We observed that the volumes of the right and left ventricular walls were altered (<5th percentile and/or >95th percentile) in 8/22 fetuses presenting with CHD. Conclusion: The reference ranges for the volumes of the right and left ventricular walls by 3DUS using STIC and VOCAL between 20w0d and 33w6d of gestation were determinate, and they exhibited good intra-observer concordance.