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In radiation therapy, fiducial markers are often implanted near tumors and used for patient positioning and respiratory gating purposes. These markers are then used to manually align the patients by matching the markers in the cone beam computed tomography (CBCT) reconstruction to those in the planning CT. This step is time-intensive and user-dependent, and often results in a suboptimal patient setup. We propose a fully automated, robust method based on dynamic programming (DP) for segmenting radiopaque fiducial markers in CBCT projection images, which are then used to automatically optimize the treatment couch position and/or gating window bounds. The mean of the absolute 2D segmentation error of our DP algorithm is mm for 87 markers on 39 patients. Intrafraction images were acquired every 3 s during treatment at two different institutions. For gated patients from Institution A (8 patients, 40 fractions), the DP algorithm increased the delivery accuracy ( versus , p  <  0.01) compared to the manual setup using kV fluoroscopy. For non-gated patients from Institution B (6 patients, 16 fractions), the DP algorithm performed similarly ( mm versus mm, p  =  0.48) compared to the manual setup matching the fiducial markers in the CBCT to the mean position. Our proposed automated patient setup algorithm only takes 1–2 s to run, requires no user intervention, and performs as well as or better than the current clinical setup. [ABSTRACT FROM AUTHOR]