Gait parameters, such as step length and step time, allow the quantification of gait deviations in persons with various diseases. Treatment can be customized and evaluated based on these parameters. However, few lowcost, easily applicable systems are available for clinical settings to accurately measure gait parameters. A lowcost spatiotemporal gait analysis system was developed and this study evaluated its accuracy and reliability. The spatiotemporal gait analysis system consists of a camera placed perpendicular to the walkway, which can be used stationary or moved manually along a parallel rail system to capture multiple strides of an individual during a single walk. Thirty-three healthy adults completed trials of barefoot, toe and shod walking. These adults were simultaneously recorded using an electronic walkway, the GAITRite®, for comparison. The results showed that the spatiotemporal gait analysis system is an accurate and reliable system to assess step and stride length, step, stance, and stride time, but not to assess double support and swing time. 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.