No data are available to describe six-degree-of-freedom (6-DOF) knee-joint kinematics for one complete cycle of overground walking following total knee arthroplasty (TKA). The aims of this study were firstly, to measure 6-DOF knee-joint kinematics and condylar motion for overground walking following TKA; and secondly, to determine whether such data differed between overground and treadmill gait when participants walked at the same speed during both tasks. A unique mobile biplane X-ray imaging system enabled accurate measurement of 6-DOF TKA knee kinematics during overground walking by simultaneously tracking and imaging the joint. The largest rotations occurred for flexion-extension and internal-external rotation whereas the largest translations were associated with joint distraction and anterior-posterior drawer. Strong associations were found between flexion-extension and adduction-abduction (R2  = 0.92), joint distraction (R2  = 1.00), and anterior-posterior translation (R2  = 0.77), providing evidence of kinematic coupling in the TKA knee. Although the measured kinematic profiles for overground walking were grossly similar to those for treadmill walking, several statistically significant differences were observed between the two conditions with respect to temporo-spatial parameters, 6-DOF knee-joint kinematics, and condylar contact locations and sliding. Thus, caution is advised when making recommendations regarding knee implant performance based on treadmill-measured knee-joint kinematic data. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1634-1643, 2017.