The convenience of the motor-driven treadmill makes it an attractive instrument for investigating rat locomotion. However, no data are available to indicate whether hindlimb treadmill kinematic findings may be compared or generalized to overground locomotion. In this investigation, we compared overground and treadmill locomotion for differences in the two-dimensional angular kinematics and temporal and spatial measurements for the hindlimb. Ten female rats were evaluated at the same speed for natural overground and treadmill walking. The walking velocity, swing duration and stride length were statistically indistinguishable between the two testing conditions. Significant differences were found between overground and treadmill locomotion for step cycle duration and stance phase duration parameters. During the stance phase of walking, the angular movement of the hip, knee and ankle joints were significantly different in the two conditions, with greater flexion occurring on the overground. Despite this, the sagittal joint movements of the hindlimb were similar between the two walking conditions, with only three parameters being significantly different in the swing. Hip height and angle-angle cyclograms were also only found to display subtle differences. This study suggests that reliable kinematic measurements can be obtained from the treadmill gait analysis in rats.