Spatiotemporal characteristics of gait such as step time and length are often associated with overall physical function in clinical populations, but can be difficult, time consuming and obtrusive to measure. This study assessed the concurrent validity of overground walking spatiotemporal data recorded using a criterion reference - a marker-based three-dimensional motion analysis (3DMA) system - and a low-cost, markerless alternative, the automated skeleton tracking output from the Microsoft Kinect™ (Kinect). Twenty-one healthy adults performed normal walking trials while being monitored using both systems. The outcome measures of gait speed, step length and time, stride length and time and peak foot swing velocity were derived using supervised automated analysis. To assess the agreement between the Kinect and 3DMA devices, Bland-Altman 95% bias and limits of agreement, percentage error, relative agreement (Pearson's correlation coefficients: r) overall agreement (concordance correlation coefficients: rc) and landmark location linearity as a function of distance from the sensor were determined. Gait speed, step length and stride length from the two devices possessed excellent agreement (r and rc values >0.90). Foot swing velocity possessed excellent relative (r=0.93) but only modest overall (rc=0.54) agreement. Step time (r=0.82 and rc=0.23) and stride time (r=0.69 and rc=0.14) possessed excellent and modest relative agreement respectively but poor overall agreement. Landmark location linearity was excellent (R(2)=0.991). This widely available, low-cost and portable system could provide clinicians with significant advantages for assessing some spatiotemporal gait parameters. However, caution must be taken when choosing outcome variables as some commonly reported variables cannot be accurately measured.