Doppler ultrasound is a noninvasive modality for portal hemodynamic study. However, inter-observer variability has been observed. This study has investigated ways to produce less inter-observer variability. Doppler ultrasound portal vein hemodynamic studies were carried out by three well-trained specialists on 20 healthy hospital staff members. The intra-hepatic, first branch, right portal vein, the hilar portal vein, and the extra-hepatic portal vein were chosen for study. With respect to the diameter of portal veins, a significant inter-observer variability was found for the first branch right portal vein and the extra-hepatic portal vein, but not for the hilar portal vein. For maximal portal vein velocity studies, inter-observer variability was not found at any location. A significant failure rate was noted for the measurement of extra-hepatic portal vein velocity. Only 8 volunteers had complete data from all of the three investigators. A significant variability was also noted for the average velocity of extra-hepatic portal vein. We conclude that Doppler ultrasound hemodynamic studies of the hilar portal vein has the most acceptable inter-observer variability and thus should be used for longitudinal portal hemodynamic studies.