To determine the inter-observer variability of defining the prostate gland on cone beam computerised tomography images for the purposes of image-guided radiotherapy.Five genitourinary oncologists contoured the prostate gland on five cone beam computerised tomography datasets. The variations in prostate boundary delineation and consequent isocentre placement between observers were measured. Variations in volume and centre of mass were calculated. The variation in boundary definition was determined with finite element modelling.The average standard deviation for centre of mass displacements was small, measuring 0.7, 1.8 and 2.8mm in the left–right, anterior–posterior and superior–inferior directions, respectively. The standard deviation for volume determination was 8.93cm 3 with large variability (3.98–19.00cm 3 ). The mean difference between the computerised tomography-derived volume and the mean cone beam-derived volume was 16% (range 0–23.7%). The mean standard deviations for left–right, anterior–posterior and superior–inferior boundary displacements were, respectively, 1.8, 2.1 and 3.6mm. The maximum deviation seen was 9.7mm in the superior direction.Expert observers had difficulty agreeing upon the location of the prostate peri-prostatic interface on the images provided. The effect on the centre of mass determination was small, and inter-observer variability for prostate detection on cone beam computerised tomography images is not prohibitive to the use of soft tissue guidance protocols. Potential exists for significant systematic matching errors, and points to the need for rigorous therapist image recognition training and development of guidance protocols before clinical implementation of soft tissue cone beam image guidance.