BACKGROUND AND PURPOSE: In-room cone-beam CT (CBCT) imaging and adaptive treatment strategies are promising methods to decrease target volumes and to spare organs at risk. The aim of this work was to analyze the inter-observer contouring uncertainties of target volumes and organs at risks (oars) in localized prostate cancer radiotherapy using CBCT images. Furthermore, CBCT contouring was benchmarked against other image modalities (CT, MR) and the influence of subjective image quality perception on inter-observer variability was assessed. METHODS AND MATERIALS: Eight prostate cancer patients were selected. Seven radiation oncologists contoured target volumes and oars on CT, MRI and CBCT. Volumes, coefficient of variation (COV), conformity index (cigen), and coordinates of center-of-mass (COM) were calculated for each patient and image modality. Reliability analysis was performed for the support of the reported findings. Subjective perception of image quality was assessed via a ten-scored visual analog scale (VAS). RESULTS: The median volume for prostate was larger on CT compared to MRI and CBCT images. The inter-observer variation for prostate was larger on CBCT (CIgen=0.57±0.09, 0.61 reliability) compared to CT (CIgen=0.72±0.07, 0.83 reliability) and MRI (CIgen=0.66±0.12, 0.87 reliability). On all image modalities values of the intra-observer reliability coefficient (0.97 for CT, 0.99 for MR and 0.94 for CBCT) indicated high reproducibility of results. For all patients the root mean square (RMS) of the inter-observer standard deviation (σ) of the COM was largest on CBCT with σ(x)=0.4 mm, σ(y)=1.1 mm, and σ(z)=1.7 mm. The concordance in delineating OARs was much stronger than for target volumes, with average CIgen>0.70 for rectum and CIgen>0.80 for bladder. Positive correlations between CIgen and VAS score of the image quality were observed for the prostate, seminal vesicles and rectum. CONCLUSIONS: Inter-observer variability for target volume delineation in prostate cancer is larger for CBCT-based contouring compared to CT and MRI. This factor of influence needs to be considered when defining safety margins for CBCT-based Adaptive Radiotherapy (ART).