Kinematic analysis is a useful and widespread tool used in research and clinical biomechanics for the quantification of human movement. Common marker-based optical motion capture systems are time intensive and require highly trained operators to obtain kinematic data. Markerless motion capture systems offer an alternative method for the measurement of kinematic data with several practical benefits. This work compared the kinematics of human gait measured using a deep learning algorithm-based markerless motion capture system to those from a standard marker-based motion capture system. Thirty healthy adult participants walked on a treadmill while data were simultaneously recorded using eight video cameras and seven infrared optical motion capture cameras, providing synchronized markerless and marker-based data for comparison. The average root mean square distance (RMSD) between corresponding joint centers was less than 2.5 cm for all joints except the hip, which was 3.6 cm. Lower limb segment angles relative to the global coordinate system indicated the global segment pose estimates from both systems were very similar, with RMSD of less than 5.5° for all segment angles except those that represent rotations about the long axis of the segment. Lower limb joint angles captured similar patterns for flexion/extension at all joints, ab/adduction at the knee and hip, and toe-in/toe-out at the ankle. These findings indicate that the markerless system would be a suitable alternative technology in cases where the practical benefits of markerless data collection are preferred.