The purpose of our study was to compare interfractional bony setup variations in pelvic anatomy with two immobilization devices, the patient-specific Vac-Lok and the generic Dual Leg Positioner system (both Civco Medical Solutions, Kalona, IA), for bilateral proton radiotherapy of the prostate. Two groups of 10 patients were studied. Computed tomography (CT) was performed three times a week, yielding 233 CT image sets for the vacuum system group and 252 for the other group. The translational shifts of the pelvic bone and prostate and rotation of the upper femurs of the femoral heads with respect to the simulation CT images were analyzed. Along the anterior-posterior and lateral axes, mean and systematic translational variations of the pelvic bone and prostate, relative to skin fiducials, were significantly lower in the Vac-Lok group (all p < 0.01) than in the Dual Leg Positioner group. Abduction of the upper femur, the dominant rotation, had random rotational variations of 1.9˚ and 2.0˚ and systematic rotations of 3.1˚ and 2.9˚ for the vacuum and generic system groups, respectively. Femoral abduction was highly correlated with anterior prostate displacement for both femurs in both groups (p < 0.01). We conclude that image guidance may be needed to correct systematic translation introduced during simulation CT, particularly with the generic immobilization system. High degrees of femoral rotation may introduce prostate translation and distal misalignment of lateral proton beams with the prostate.