The aim of this work was to analyze interfraction and intrafraction deviations and residual set-up errors (RSE) after online repositioning to determine PTV margins for 3different alignment techniques in prostate cancer radiotherapy. The present prospective study included 44prostate cancer patients with implanted fiducials treated with three-dimensional (3D) conformal radiotherapy. Daily localization was based on skin marks followed by marker detection using kilovoltage (kV) imaging and subsequent patient repositioning. Additionally, in-treatment megavoltage (MV) images were obtained for each treatment field. In an off-line analysis of 7,273images, interfraction prostate motion, RSE after marker-based prostate localization, prostate position during each treatment session, and the effect of treatment time on intrafraction deviations were analyzed to evaluate PTV margins. Margins accounting for interfraction deviation, RSE and intrafraction motion were 14.1, 12.9, and 15.1mm in anterior-posterior (AP), superior-inferior (SI), and left-right (LR) direction for skin mark alignment and 9.6, 8.7, and 2.6mm for bony structure alignment, respectively. Alignment to implanted markers required margins of 4.6, 2.8, and 2.5mm. As margins to account for intrafraction motion increased with treatment prolongation PTV margins could be reduced to 3.9, 2.6, and 2.4mm if treatment time was ≤ 4min. With daily online correction and repositioning based on implanted fiducials, a significant reduction of PTV margins can be achieved. The use of an optimized workflow with faster treatment techniques such as volumetric modulated arc techniques (VMAT) could allow for a further decrease.