Original ArticleAbstract Purpose: Various factors cause geometric uncertainties during prostate radiotherapy, including interfractional and intrafractional patient motions, organ motion, and daily setup errors. This may lead to increased normal tissue complications when a high dose to the prostate is administered. More-accurate treatment delivery is possible with daily imaging and localization of the prostate. This study aims to measure the shift of the prostate by using kilovoltage (kV) cone beam computed tomography (CBCT) after position verification by kV orthogonal portal imaging (OPI). Methods: Position verification in 10 patients with prostate cancer was performed by using OPI followed by CBCT before treatment delivery in 25 sessions per patient. In each session, OPI was performed by using an on-board imaging (OBI) system and pelvic bone-to-pelvic bone matching was performed. After applying the noted shift by using OPI, CBCT was performed by using the OBI system and prostate-to-prostate matching was performed. The isocenter shifts along all three translational directions in both techniques were combined into a three-dimensional (3-D) iso-displacement vector (IDV). Results: The mean (SD) IDV (in centimeters) calculated during the 250 imaging sessions was 0.931 (0.598, median 0.825) for OPI and 0.515 (336, median 0.43) for CBCT, p-value was less than 0.0001 which shows extremely statistical significant difference. Conclusion: Even after bone-to-bone matching by using OPI, a significant shift in prostate was observed on CBCT. This study concludes that imaging with CBCT provides a more accurate prostate localization than the OPI technique. Hence, CBCT should be chosen as the preferred imaging technique.