Computer-assisted treatments have become increasingly common. Consequently, there is an increased desire for navigation methods with simplified workflow. Anatomic-based pair-point registration is often mentioned as a source of error. Alternatively, the use of preoperatively implanted markers for registration remains complex. The self-acting registration of Iso-C-3D at the moment of data acquisition can reduce essential errors. The aim of this study was to evaluate the effect of reference placement on accuracy, and to determine the maximum acceptable distance between the reference and a given isocentre. This study demonstrates the interdependence of the reference distance on the region of interest (ROI). The mean error of registration amounts to 0.04 mm (0.04-0.05 mm) up to a distance of 200 mm and beyond 0.25 mm (0.24-0.26) for distances beyond 200 mm. The accuracy was significantly lower (p<0.0001) with a distance more than 200 mm. For optimal accuracy when utilizing navigation for pelvic and long bone surgery, the reference base should not been placed at a distance more than 200 mm from the isocentre of interest.