Accurate measurement and analysis of 3D kinematics of temporomandibular joint (TMJ) is very important for assisting clinical diagnosis and treatment of prosthodontics and orthodontics, and oral surgery. This study presents a new 3D kinematic measurement technique of the TMJ using X-ray fluoroscopic images, which can easily obtain the TMJ kinematic data in natural motion. In vivo kinematics of the TMJ (maxilla and mandibular bone) is determined using a feature-based 2D/3D registration, which uses beads silhouette on fluoroscopic images and 3D surface bone models with beads. The 3D surface models of maxilla and mandibular bone with beads were created from CT scans data of the subject using the mouthpiece with the seven strategically placed beads. In order to validate the accuracy of pose estimation for the maxilla and mandibular bone, computer simulation test was performed using five patterns of synthetic tantalum beads silhouette images. In the clinical applications, dynamic movement during jaw opening and closing was conducted, and the relative pose of the mandibular bone with respect to the maxilla bone was determined. The results of computer simulation test showed that the root mean square errors were sufficiently smaller than 1.0 mm and 1.0 degree. In the results of clinical application, during jaw opening from 0.0 to 36.8 degree of rotation, mandibular condyle exhibited 19.8 mm of anterior sliding relative to maxillary articular fossa, and these measurement values were clinically similar to the previous reports. Consequently, present technique was thought to be suitable for the 3D TMJ kinematic analysis.