Most X-ray fluoroscopy systems are stationary and impose restrictions on the measurement of dynamic joint motion; for example, knee-joint kinematics during gait is usually measured with the subject ambulating on a treadmill. We developed a computer-controlled, mobile, biplane, X-ray fluoroscopy system to track human body movement for high-speed imaging of 3D joint motion during overground gait. A robotic gantry mechanism translates the two X-ray units alongside the subject, tracking and imaging the joint of interest as the subject moves. The main aim of the present study was to determine the accuracy with which the mobile imaging system measures 3D knee-joint kinematics during walking. In vitro experiments were performed to measure the relative positions of the tibia and femur in an intact human cadaver knee and of the tibial and femoral components of a total knee arthroplasty (TKA) implant during simulated overground gait. Accuracy was determined by calculating mean, standard deviation and root-mean-squared errors from differences between kinematic measurements obtained using volumetric models of the bones and TKA components and reference measurements obtained from metal beads embedded in the bones. Measurement accuracy was enhanced by the ability to track and image the joint concurrently. Maximum root-mean-squared errors were 0.33 mm and 0.65° for translations and rotations of the TKA knee and 0.78 mm and 0.77° for translations and rotations of the intact knee, which are comparable to results reported for treadmill walking using stationary biplane systems. System capability for in vivo joint motion measurement was also demonstrated for overground gait.