Background and Purpose: : In order to use a single implant with one treatment plan in fractionated high-dose-rate brachytherapy (HDR-B), applicator position shifts must be corrected prior to each fraction. The authors investigated the use of gold markers for X-ray-based setup and position control between the single fractions. Patients and Methods: : Caudad-cephalad movement of the applicators prior to each HDR-B fraction was determined on radiographs using two to three gold markers, which had been inserted into the prostate as intraprostatic reference, and one to two radiopaque-labeled reference applicators. 35 prostate cancer patients, treated by HDR-B as a monotherapy between 10/2003 and 06/2006 with four fractions of 9.5 Gy each, were analyzed. Toxicity was scored according to the CTCAE Score, version 3.0. Median follow-up was 3 years. Results: : The mean change of applicators positions compared to baseline varied substantially between HDR-B fractions, being 1.4 mm before fraction 1 (range, -4 to 2 mm), -13.1 mm before fraction 2 (range, -36 to 0 mm), -4.1 mm before fraction 3 (range, -21 to 9 mm), and -2.6 mm at fraction 4 (range, -16 to 9 mm). The original position of the applicators could be readjusted easily prior to each fraction in every patient. In 18 patients (51%), the applicators were at least once readjusted > 10 mm, however, acute or late grade ≥ 2 genitourinary toxicity was not increased (p = 1.0) in these patients. Conclusion: : Caudad position shifts up to 36 mm were observed. Gold markers represent a valuable tool to ensure setup accuracy and precise dose delivery in fractionated HDR-B monotherapy of prostate cance