BACKGROUND In-room cone-beam computerized tomography (CBCT) imaging is a promising method to reduce setup errors, especially in organs such as the bladder that often have large intrafractional variations due to organ movement. CBCT image quality is limited by low contrast and imaging artifacts, but few data have been reported about inter-observer variability of bladder boundary delineation on CBCT. The aim of this work was to analyze and evaluate the inter-observer contouring uncertainties of bladder boundary delineation on CBCT images in a prospective fashion. METHODS Five radiation oncologists contoured 10 bladders using the CBCT datasets of consecutive 10 patients (including 4 females) who were irradiated to the pelvic region. Prostates were also contoured in male patients. Patients who had had prostatectomy were excluded. The coefficient of variation (COV), conformity index (CI(gen)), and coordinates of center-of-mass (COM) of the bladder and prostate were calculated for each patient. RESULTS The mean COV for the bladder and prostate was 0.08 and 0.20, respectively. The mean CI(gen) of the bladder and prostate was 0.81 and 0.66, respectively. The root mean square (RMS) of the inter-observer standard deviation (σ) of the COM displacement in the left-right (LR) and anterior-posterior (AP) direction was 0.79, 0.87 and 0.54 for the bladder and 0.63, 0.99 and 1.72 for the prostate. Regarding the mean COV and CI(gen) for the bladder, the differences between males and females were not significant. CONCLUSIONS Inter-observer variability for bladder delineation on CBCT images was substantially small regardless of gender. We believe that our results support the applicability of CBCT in adaptive radiotherapy for bladder cancer.