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Background: The aim of this study is to compare three methodologies of prostate localization and to determine if there are significant differences in the techniques., Methods: Daily prostate localization using cone beam CT or orthogonal kV imaging has been performed at UT Southwestern Medical Center since 2006. Prostate patients are implanted with gold seeds, which are matched with the planning CT or DRR before treatment. More recently, a technology using electromagnetic transponders implanted within the prostate was introduced into our clinic (Calypso®). With each technology, patients are localized initially using skin marks and the room lasers. In this study, patients were localized with Calypso and either CBCT or kV orthogonal images in the same treatment session, allowing a direct comparison of the technologies. Localization difference distributions were determined from the difference in the offsets determined by CBCT/kV imaging and Calypso. CBCT-Calypso and kV imaging-Calypso localization data were summarized from over 900 and 250 fractions each, respectively. The Wilcoxon signed rank test is used to determine if the localization differences are statistically significant. We also calculated Pearson's product-moment correlation coefficient (R2) to determine if there is a linear relationship between the shifts determined by Calypso and the radiographic techniques., Results: The differences between CBCT-Calypso and kV imaging-Calypso localizations are -0.18 ± 2.90 mm, -0.79 ± 2.18 mm, -0.01 ± 1.20 mm and -0.09 ± 1.40 mm, 0.48 ± 1.50 mm, 0.08 ± 1.04 mm, respectively, in the AP, SI, and RL directions. The Pearson product-moment correlation coefficients for the CBCT-Calypso shifts were 0.71, 0.92 and 0.88 and for the OBI-Calypso comparison were 0.95, 0.89 and 0.85. The percentage of localization differences that were less than 3 mm were 86.1%, 84.5% and 96.0% for the CBCT-Calypso comparison and 95.8%, 94.3% and 97% for the kV OBI-Calypso comparison. No trends were observed in the Bland-Altman analysis., Conclusions: Localization of the prostate using electromagnetic transponders agrees well with radiographic techniques and each technology is suitable for high precision radiotherapy. This study finds that there is more uncertainty in CBCT localization of the prostate than in 2D orthogonal imaging, but the difference is not clinically significant.