Objective: To determine the concurrent validity and reliability of a low-cost spatiotemporal gait analysis system for clinical use in rehabilitation medicine. Design: Cross-sectional study. Subjects: Thirty-three healthy adults. Methods: The spatiotemporal gait analysis system consists of a video camera placed perpendicular to a 10-m walkway and calibrated for spatial reference. The conditions evaluated in this study were: barefoot walking at comfortable and slow speed, toe and shod walking using a stationary camera setup and barefoot walking at comfortable speed using a moving camera setup. The GAITRite® was used as reference. Results: High intraclass correlation coefficients (ICC≥0.97; 95% lower limit confidence intervals (95% CIs) ≥0.77) were found between systems for step and stride length, and step, stance and stride time, across setups and conditions. Standard error of measurement and Bland-Altman repeatability coefficients were ≤2.4% and ≤6.3%, respectively. A minimum of 4 footsteps was required to obtain ICC >0.90 and coefficient of variation <10%. For double support and swing time, ICCs were generally low (ICC≥0.21). Inter-rater reliability was excellent for step length, step and stance time (ICC≥0.94; lower limit 95% CIs ≥0.86). Conclusion: The spatiotemporal gait analysis system is valid and reliable for assessing spatiotemporal parameters in different walking conditions. However, the validity of double support and swing time could not be confirmed.