Vision-based methods using RGB inputs for human pose estimation have grown in recent years but have undergone limited testing in clinical and biomechanics research areas like gait analysis. The purpose of the present study was to compare lower extremity kinematics during overground gait between a traditional marker-based approach and a commercial multi-view markerless system in a sample of subjects including young adults, older adults, and adults diagnosed with Parkinson’s disease. A convenience sample of 35 adults between the age of 18–85 years were included in this study, yielding a total of 114 trials and 228 gait cycles that were compared between systems. A total of 30 time normalized waveforms, including three-dimensional joint centers, segment angles, and joint angles were compared between systems using root mean-squared error (RMSE), range of motion difference (ΔROM), Pearson correlation coefficients (r), and interclass correlation coefficients (ICC). RMSEs for joint center positions were less than 28 mm in all joints with correlations indicating good to excellent agreement. RMSEs for segment and joint angles were in range of previous results, with highest agreement between systems in the sagittal plane. ΔROM differences were within reference values that characterize clinical groups like Parkinson’s disease, stroke, or knee osteoarthritis. Further improvements in pelvis tracking, markerless keypoint model definitions, and standardization of comparison study protocols are needed. Nevertheless, markerless solutions seem promising toward unrestricted motion analysis in biomechanics research and clinical settings.