BACKGROUND: To analyze interfraction motion of seminal vesicles (SV), and its motion relative to rectal and bladder filling. METHODS AND MATERIALS: SV and prostate were contoured on 771 daily computed tomography "on rails" scans from 24 prostate cancer patients undergoing radiotherapy. Random and systematic errors for SV centroid displacement were measured relative to the prostate centroid. Margins required for complete geometric coverage of SV were determined using isotropic expansion of reference contours. SV motion relative to rectum and bladder was determined. RESULTS: Systematic error for the SV was 1.9 mm left-right (LR), 2.9 mm anterior-posterior (AP) and 3.6 mm superior-inferior (SI). Random error was 1.4 mm (LR), 2.7 mm (AP) and 2.1 mm (SI). 10 mm margins covered the entire left SV and right SV on at least 90% of fractions in 50% and 33% of patients and 15 mm margins covered 88% and 79% respectively. SV AP movement correlated with movement of the most posterior point of the bladder (mean R2 = 0.46, SD = 0.24) and rectal area (mean R2 = 0.38, SD = 0.21). CONCLUSIONS: Considerable interfraction displacement of SV was observed in this cohort of patients. Bladder and rectal parameters correlated with SV movement.