The treadmill is an attractive device for the investigation of human locomotion, yet the extent to which lower limb kinematics differ from overground running remains a controversial topic. This study aimed to provide an extensive three-dimensional kinematic comparison of the lower extremities during overground and treadmill running. Twelve participants ran at 4.0 m/s (+/- 5%) in both treadmill and overground conditions. Angular kinematic parameters of the lower extremities during the stance phase were collected at 250 Hz using an eight-camera motion analysis system. Hip, knee, and ankle joint kinematics were quantified in the sagittal, coronal, and transverse planes, and contrasted using paired t-tests. Of the analysed parameters hip flexion at footstrike and ankle excursion to peak angle were found to be significantly reduced during treadmill running by 12 degrees (p = 0.001) and 6.6 degrees (p = 0.010), respectively. Treadmill running was found to be associated with significantly greater peak ankle eversion (by 6.3 degrees, p = 0.006). It was concluded that the mechanics of treadmill running cannot be generalized to overground running.