Clinical evaluation of an optical three-dimensional surface scanning (3D-SS) system for patient positioning and monitoring during radiotherapy (RT) for breast cancer. A ceiling-mounted scanner was developed to acquire multiple 3D body surface images and tested in 14 conservatively operated breast cancer patients. A reference skin surface was derived from the planning computed tomography (CT) scan as basis for rigid registration with the surface scans. In addition to electronic portal images (EPIs), optical scans were acquired at three defined time points before and during daily RT. Patient setup was guided by laser alignments and corrected according to EPI findings. The accuracy of the 3D-SS system was validated by comparison of the optical scans to EPIs generated in parallel. Interfraction shifts were investigated by comparison of the first 3D-SS image with the reference body outline. Intrafractional motions were analysed by comparing the three daily surface scans with the first EPI. Comparison of EPIs and 3D-SS images revealed good accordance (- 0.05±0.94 mm). Analysis of daily patient positions revealed average deviations of 0.4±2.4 mm laterally, 0.3±1.9 mm longitudinally and 0.2±3.3 mm vertically. After 2 weeks, a systematic interfraction shift in patient positioning was noted, particularly in the vertical direction (4.9±0.56 mm), which was attributed to patients progressively relaxing. 3D-SS images showed intrafractional shifts of 1.2±0.7 mm over a time course of 2 min. Optical surface scanning is a simple, fast and reproducible method for breast cancer patient alignment. Particularly for more sophisticated irradiation techniques, it helps to improve accuracy in patient positioning during radiotherapy without the exposure to additional ionizing radiation.