Objectives: To determine Z-score equations and reference ranges for mitral valve-tricuspid valve distance (MTD) and the MTD index in the fetal heart. Methods: A prospective cross-sectional study was performed in 899 normal singleton fetuses from 14 to 40 weeks’ gestation. The MTD and interventricular septum length (IVSL) were measured offline after electronic cardiac spatiotemporal image correlation volume acquisition. The MTD index was determined as the ratio of MTD to IVSL. Z-score reference ranges of these measurements were determined against gestational age (GA) and estimated fetal weight (EFW), using regression analysis of the mean and standard deviation (SD). Results: Strong positive correlations were found between the MTD and the independent variables. A simple linear regression model was the best description of the mean and SD of MTD based on GA, while a cubic regression best fitted the mean MTD against EFW. In contrast, the MTD index decreased progressively with the independent variables. Fractional polynomials best fitted the MTD index in terms of GA and EFW. Conclusion: Normal reference values and Z-scores of fetal MTD and MTD index were provided against GA and EFW, which may be useful tools for quantitative assessment of some cardiac and extracardiac diseases.