Assessment of fetal cardiac function is difficult because of limited accessibility. Tissue Doppler imaging (TDI) is a promising technique in assessing diastolic function in adults. There has been sparseness concerning the use of TDI in assessing fetal cardiac function. The aim of this study was to construct reference charts of TDI indices of systolic and diastolic functions of the normal fetal heart. Ventricular myocardial velocities at the left ventricular (LV) wall, right ventricular (RV) wall, and interventricular septum (IVS) were assessed by TDI in 302 subjects. From 19 to 37 weeks of gestation, peak myocardial velocities during early diastole (Em) increased from 3.3 to 7.2, 3.9 to 8.3, and 3.2 to 5.0 m/s at the LV wall, RV wall, and IVS, respectively. Peak myocardial velocities during atrial contraction (Am) also increased throughout gestation, but the magnitude of increase was smaller (6.3 to 7.9, 7.7 to 10.6, and 5.5 to 5.9 m/s for the LV wall, RV wall, and IVS, respectively). As a consequence, the Em/Am ratio increased from 0.51 to 0.61 at midtrimester to 0.76 to 0.91 at term. Similar to Em, peak myocardial velocities during systole (Sm) also increased by almost 2 times from 18 to 37 weeks of gestation (3.8 to 6.0, 4.2 to 7.6, and 3.3 to 5.6 for the LV wall, RV wall, and IVS, respectively). Reference charts of gestation-specific Em, Am, Em/Am ratio, Sm, and E/Em were constructed accordingly. In midtrimester, fetal diastolic function is predominantly contributed by atrial contraction. As gestation advances, ventricular relaxation becomes increasingly mature. Reference charts for TDI indices were constructed, these will allow identification of fetuses with an abnormal diastolic function.