A comparison of six similarity measures for use in intensity based 2D-3D image registration is presented. The accuracy of the similarity measures are compared to a “gold-standard” registration which has been accurately calculated using fiducial markers. The similarity measures are used to register a CT scan to a fluoroscopy image of a spine phantom. The registration is carried out within a region of interest in the fluoroscopy image which is user defined to contain a single vertebra. Many of the problems involved in this type of registration are caused by features which were not modelled by a phantom image alone. More realistic “gold standard” data sets were simulated using the phantom image with clinical image features overlaid. Results show that the introduction of soft tissue structures and interventional instruments into the phantom image can have a large effect on the performance of some similarity measures previously applied to 2D-3D image registration. Two measures were able to register accurately and robustly even when soft tissue structures and interventional instruments were present as differences between the images. These measures are called pattern intensity and gradient difference.