Radiographic data, including computed tomography (CT) and planar X-ray, is increasingly used for human and animal kinematic studies. There is a tendency toward using as high-resolution imaging as possible. Higher resolution imaging is one factor (in conjunction with the reconstruction algorithm), which may increase the precision of reconstructed three-dimensional (3D) surface models in representing true bone shape. However, to date no study has tested the effects of scan resolution, threshold, and 3D model reconstruction algorithm on the accuracy of bone kinematic results. The present study uses a novel method to do this where canine tarsal bones were positioned on a radiolucent Lego™ board and scanned before and after undergoing known translations and/or rotations. The digital imaging and communications in medicine (DICOM) images were acquired using two different CT scanning resolutions and processed using three different segmentation threshold levels and three different reconstruction algorithms. Using one bone as the reference bone, an iterative closest point (ICP) algorithm was used to register bones to a global co-ordinate system and allow measurement of other bone kinematics in terms of translations and rotations in and around the x-, y-, and z-axes. The measured kinematics were compared to the “known” kinematics, which were obtained from the Lego™ board's manufacturing standards and tolerances, to give accuracy error metrics for all bones. The results showed error in accuracy of measured kinematics was at subvoxel levels (less than 0.5 mm). Despite altering the volume and surface area of the 3D bone models, variation in resolution, segmentation threshold and reconstruction algorithm had no significant influence upon the accuracy of the calculated tarsal bone kinematics.