Spatiotemporal parameters can characterize the gait patterns of individuals, allowing assessment of their health status and detection of clinically meaningful changes in their gait. Video-based markerless motion capture is a user-friendly, inexpensive, and widely applicable technology that could reduce the barriers to measuring spatiotemporal gait parameters in clinical and more diverse settings. The aim of this work was to determine whether spatiotemporal gait parameters measured using Theia3D markerless motion capture demonstrate concurrent validity with those measured using marker-based motion capture. Thirty healthy adult participants performed treadmill walking at self-selected speeds while 2D video and marker-based motion capture data were collected simultaneously. Kinematic-based gait events were used to measure nine spatiotemporal gait parameters from both systems independently. The parameters were compared using their group means, Bland-Altman methods, Pearson correlation coefficients, paired-samples t-tests, and intraclass correlation coefficients (ICC(A-1) and ICC(C-1)). Group means between systems were indistinguishable across all nine gait parameters, and the Bland-Altman plots showed no systematic biases or clinically meaningful differences between the systems. Pearson coefficients indicated near-perfect correlations (r ≥0.96) between systems for all but two parameters, which had strong correlations (double-limb support time, r=0.87; swing time, r=0.88). T-tests indicated differences in stance, swing, and double-limb support time parameters, but strong correlations were found for all ICCs, the lowest being 0.84 for both double-limb support time and swing time. The measurements made by the Theia3D markerless motion capture system demonstrated concurrent validity with those from the marker-based system, indicating sufficient accuracy for research, clinical, and other uses.