Objective: To evaluate interfractional and intrafractional movement of patients with rectal cancer during radiotherapy with electronic portal imaging device (EPID) and surface infrared (IR) markers. Methods: 20 patients undergoing radiotherapy for rectal cancer with body mass index ranging from 18.5 to 30 were enrolled. Patients were placed in the prone position on a couch with a leg pillow. Three IR markers were put on the surface of each patient and traced by two stereo cameras during radiotherapy on a twice-weekly basis. Interfractional isocentre movement was obtained with EPID images on a weekly basis. Movement of the IR markers was analysed in correlation with the isocentre movement obtained from the EPID images. Results: The maximum right-to-left (R-L) movement of the laterally located markers in the horizontal isocentre plane was correlated with isocentre translocation with statistical significance (p50.018 and 0.015, respectively). Movement of the surface markers was cyclical. For centrally located markers, the 95% confidence intervals for the average amplitude in the R-L, cranial-to-caudal (C-C) and anterior-to-posterior (A-P) directions were 0.86, 2.25 and 3.48 mm, respectively. In 10 patients, intrafractional movement exceeding 5mm in at least one direction was observed. Time-dependent systematic movement of surface markers during treatment, which consisted of continuous movement towards the cranial direction and a sail back motion in the A-P direction, was also observed. Conclusion: Intrafractional movement of surface markers has both cyclic components and time-dependent systematic components. Marker deviations exceeding 5mm were mainly seen in the A-P direction. Pre-or post-treatment EPID images may not provide adequate information regarding intrafractional movement because of systematic movement in the A-P direction during radiotherapy. Advances in knowledge: This work uncovered a sail back motion of patients in the A-P direction during radiotherapy. Pre-or post-treatment EPID images may not provide accurate positioning of patients in the A-P direction because of this time-dependent intrafractional motion.