PURPOSE: To determine intra-fraction displacement of the prostate gland from imaging pre- and post-radiotherapy delivery of prostate cancer patients with three implanted fiducial markers. METHODS AND MATERIALS: Data were collected from 184 patients who had two orthogonal X-rays pre- and post-delivery on at least 20 occasions using a Varian On Board kV Imaging system. A total of 5778 image pairs covering time intervals between 3 and 30 min between pre- and post-imaging were evaluated for intra-fraction prostate displacement. RESULTS: The mean three dimensional vector shift between images was 1.7 mm ranging from 0 to 25 mm. No preferential direction of displacement was found; however, there was an increase of prostate displacement with time between images. There was a large variation in typical shifts between patients (range 1 +/- 1 to 6 +/- 2 mm) with no apparent trends throughout the treatment course. Images acquired in the first five fractions of treatment could be used to predict displacement patterns for individual patients. CONCLUSION: Intra-fraction motion of the prostate gland appears to be a limiting factor when considering margins for radiotherapy. Given the variation between patients, a uniform set of margins for all patients may not be satisfactory when high target doses are to be delivered.