The procedure required for registering a surgical navigation system prior to use in a surgical procedure is conventionally a time-consuming manual process that is prone to human errors and must be repeated as necessary through the course of a procedure. The conventional procedure becomes even more time consuming when intra-operative 3D imaging such as the C-arm cone-beam CT (CBCT) is introduced, as each updated volume set requires a new registration. To improve the speed and accuracy of registering image and world reference frames in image-guided surgery, a novel automatic registration algorithm was developed and investigated. The surgical navigation system consists of either Polaris (Northern Digital Inc., Waterloo, ON) or Micron Tracker (Claron Technology Inc., Toronto, ON) tracking camera(s), custom software (Cogito running on a PC), and a prototype CBCT imaging system based on a mobile isocentric C-arm (Siemens, Erlangen, Germany). Experiments were conducted to test the accuracy of automatic registration methods for both the Micron Tracker and Polaris tracking cameras. Results indicate the automated registration performs as well as the manual registration procedure using either the Claron or Polaris camera. The average root-mean-squared (rms) observed target registration error (TRE) for the manual procedure was 2.58 +/-0.42 mm and 1.76 +/-0.49 mm for the Polaris and Micron Tracker, respectively. The mean observed TRE for the automatic algorithm was 2.11 +/-0.13 and 2.03 +/-0.3 mm for the Polaris and Micron Tracker, respectively. Implementation and optimization of the automatic registration technique in C-arm CBCT guidance of surgical procedures is underway.