Assessment of intraventricular pressure gradients (IVPG) using color Doppler M-mode echocardiography has gained increasing interest in the evaluation of cardiac function. However, standardized analysis tools for IVPG quantification are missing. We aimed to evaluate the feasibility, the test-retest observer reproducibility, and the inter-system variability of a semi-automated IVPG quantification algorithm. The study included forty healthy volunteers (50% were men). All volunteers were examined using two ultrasound systems, the Philips Epiq 7 and the General Electric Vivid 6. Left ventricular diastolic (DIVPG) and systolic (SIVPG) intraventricular pressure gradients were measured from the spatiotemporal distribution of intraventricular propagation flow velocities using color Doppler M-mode in standard apical views. There was good feasibility for both systolic and diastolic IVPG measurements (82.5% and 85%, respectively). Intra and inter-observer test-retest variability measured with the intraclass correlation coefficient were 0.98 and 0.93 for DIVPG respectively, and 0.95 and 0.89 for SIVPG respectively. The inter-system concordance was weak to moderate with Lin's concordance correlation coefficient of 0.59 for DIVPG and 0.25 for SIVPG. In conclusion, it is feasible and reproducible to assess systolic and diastolic IVPG using color Doppler M-mode in healthy volunteers. However, the inter-system variability in IVPG analysis needs to be taken into account, especially when using displayed data.