The purpose of this study was to compare interfraction prostate displacement data between electronic portal imaging (EPI) and kilovoltage imaging (KVI) treatment units and discuss the impact of any difference on margin calculations for prostate cancer image-guided radiotherapy (IGRT).Prostate interfraction displacement data was collected prospectively for the first 4 fractions in 333 patients treated with IGRT with daily pre-treatment EPI or KVI orthogonal imaging. Displacement was recorded in the anteroposterior (AP), left-right (LR) and superoinferior (SI) directions. The proportion of displacement <3 mm and the difference in median absolute displacements were calculated in all directions.1088 image pairs were analysed in total, 448 by EPI and 640 by KVI. There were 23% (95% confidence interval [CI] 18-28%) more displacements under 3 mm for EPI than for KVI in the AP direction, 14% (95% CI 10-19%) more in the LR direction and 10% (95% CI 5-15%) more in the SI direction. The differences in absolute median displacement (KVI>EPI) were AP 1 mm, LR 1 mm and SI 0.5 mm. Wilcoxon rank-sum test showed that distributions were significantly different for all three dimensions (p<0.0001 for AP and LR and p=0.02 for SI).EPI has a statistically significant smaller set-up error distribution than KVI. We would expect that, because fiducial marker imaging is less clear for EPI, the clinical target volume to planning target volume margin would be greater when using IGRT; however, relying wholly on displacement data gives the opposite result. We postulate that this is owing to observer bias, which is not accounted for in margin calculation formulas.