It is thought that the cardiac chambers differ in their morphological and contractile properties from an early embryonic stage of development. We hypothesized that a non-invasive ultrasonic backscatter investigation might illustrate fundamental differences in myocardial properties of the two ventricles during heart development. The goals of this investigation were: 1) to measure the apparent level of backscatter from the left and right ventricular walls of excised, mid-gestational fetal pig hearts; 2) to measure the magnitude of the cyclic variation over the heart cycle of ultrasonic backscatter from the left and right ventricular free walls of mid-gestational fetal human hearts in utero; and 3) to determine if the observed cyclic variation of backscatter results are consistent with predictions relating overall backscatter levels and the magnitudes of cyclic variation. Broadband (30 to 60 MHz) integrated backscatter measurements were obtained from the left and right ventricular myocardium from 16 excised, formalin-fixed, mid-gestational fetal pig heart specimens using a 50-MHz single-element transducer measurement system. Separately, measurements of the magnitude of cyclic variation of backscatter from the left and right ventricular walls of the hearts of 16 structurally normal mid-gestational human fetuses were obtained from analyses of echocardiographic images obtained using a specially-configured commercial imaging system. For each of the 16 fetal pig hearts, the level of apparent integrated backscatter from the right ventricular myocardium was larger than that from the left ventricular myocardium, exhibiting mean values of -35.9 ± 2.0 dB and -40.1 ± 1.9 dB (mean ± SD; p < 0.001), respectively. For the human fetuses, the magnitude of cyclic variation of backscatter was found to be greater for the left ventricular free wall than for the right ventricular free wall (4.5 ± 1.1 dB vs. 2.3 ± 0.9 dB, respectively; mean ± SD; p < 0.0001). Measure ments of the level of apparent backscatter (fetal pigs) and cyclic variation of backscatter (human fetuses) demonstrate significant differences between the left and right ventricular myocardium. The measured results are consistent with previously described models relating overall backscatter levels and the magnitudes of cyclic variation that predict the magnitude of cyclic variation should decrease as the level of backscatter from the myocardium increases. Results of this study suggest cyclic variation measurements may offer a useful approach for characterizing intrinsic differences in myocardial properties of the two ventricles in assessing fetal heart development.