In gait analysis, knowledge on validity and reliability of instruments and influences caused by the examiner’s performance is of crucial interest. These measurement properties are not yet known for commonly used, low-cost two-dimensional (2D) video-based systems. The purpose of this study was to assess the concurrent validity of a video-based 2D system against a three-dimensional (3D) reference standard, as well as the inter-rater reliability, and test–retest reliability of 3D marker application. Level walking was captured simultaneously by a 2D and a 3D system. Reflective markers were applied independently by three raters and repeated by one rater on a second day. We assessed the agreement between the two systems, as well as reproducibility, and inter-rater agreement of derived spatio-temporal parameters and sagittal kinematics. Nineteen healthy participants completed this study. 2D gait analysis provides a possibility to accurately assess parameters such as stride time, stride length, gait velocity, and knee RoM. Interrater and test–retest reliability of 3D gait analysis are generally acceptable, except for the parameters toe-off and pelvic RoM. This is the first study to publish measurement properties of a commercially available 2D video-based gait analysis system, which can support interpretation of gait pattern near the sagittal plane.