To evaluate on-board digital tomosynthesis (DTS) for patient positioning vs. two-dimensional (2D) radiography and three-dimensional cone beam (CBCT).A total of 92 image sessions from 9 prostate cancer patients were analyzed. An on-board image set was registered to a corresponding reference image set. Four pairs of image sets were used: digitally reconstructed radiographs vs. on-board orthogonal paired radiographs for the 2D method, coronal-reference DTS vs. on-board coronal DTS for the coronal-DTS method, sagittal-reference DTS vs. on-board sagittal DTS for the sagittal-DTS method, and planning CT vs. CBCT for the CBCT method. The registration results were compared.The systematic errors in all methods were <1 mm/1 degrees . When registering the bony anatomy, the mean vector difference was 0.21 +/- 0.11 cm between 2D and CBCT, 0.11 +/- 0.08 cm between CBCT and coronal DTS, and 0.14 +/- 0.07 cm between CBCT and sagittal DTS. The correlation between CBCT to DTS was stronger (coefficient = 0.92-0.95) than the correlation between 2D and CBCT or DTS (coefficient = 0.81-0.83). When registering the soft tissue, the mean vector difference was 0.18 +/- 0.11 cm between CBCT and coronal DTS and 0.29 +/- 0.17 cm between CBCT and sagittal DTS. The correlation coefficient of CBCT to sagittal DTS and to coronal DTS was 0.84 and 0.92, respectively.DTS could provide equivalent results to CBCT when the bony anatomy is used as landmarks for prostate image-guided radiotherapy. For soft tissue-based positioning verification, coronal DTS produced equivalent results to CBCT, but sagittal DTS alone was insufficient. DTS could allow for comparable soft tissue-based target localization with faster scanning time and a lower imaging dose compared with CBCT.