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Abstract Background Intensity-modulated radiotherapy (IMRT) enables a better conformality to the target while sparing the surrounding normal tissues and potentially allows to increase the dose to the target, if this is precisely and accurately determined. The goal of this work is to determine inter-fraction setup errors and prostate motion in IMRT for localized prostate cancer, guided by daily kilovoltage cone beam computed tomography (kVCBCT). Methods Systematic and random components of the shifts were retrospectively evaluated by comparing two matching modalities (automatic bone and manual soft-tissue) between each of the 641 daily kVCBCTs (18 patients) and the planning kVCT. A simulated Adaptive Radiation Therapy (ART) protocol using the average of the first 5 kVCBCTs was tested by non-parametric bootstrapping procedure. Results Shifts were < 1 mm in left-right (LR) and in supero-inferior (SI) direction. In antero-posterior (AP) direction systematic prostate motion (2.7 ± 0.7 mm) gave the major contribution to the variability of results; the averages of the absolute total shifts were significantly larger in anterior (6.3 ± 0.2 mm) than in posterior (3.9 mm ± 0.2 mm) direction. The ART protocol would reduce margins in LR, SI and anterior but not in posterior direction. Conclusions The online soft-tissue correction based on daily kVCBCT during IMRT of prostate cancer is fast and efficient. The large random movements of prostate respect to bony anatomy, especially in the AP direction, where anisotropic margins are needed, suggest that daily kVCBCT is at the present time preferable for high dose and high gradients IMRT prostate treatments.