Background Caenorhabditis elegans locomotion is a simple behavior that has been widely used to dissect genetic components of behavior, synaptic transmission, and muscle function. Many of the paradigms that have been created to study C. elegans locomotion rely on qualitative experimenter observation. Here we report the implementation of an automated tracking system developed to quantify the locomotion of multiple individual worms in parallel.Methodology/Principal FindingsOur tracking system generates a consistent measurement of locomotion that allows direct comparison of results across experiments and experimenters and provides a standard method to share data between laboratories. The tracker utilizes a video camera attached to a zoom lens and a software package implemented in MATLAB®. We demonstrate several proof-of-principle applications for the tracker including measuring speed in the absence and presence of food and in the presence of serotonin. We further use the tracker to automatically quantify the time course of paralysis of worms exposed to aldicarb and levamisole and show that tracker performance compares favorably to data generated using a hand-scored metric.Conclusions/SignficanceAlthough this is not the first automated tracking system developed to measure C. elegans locomotion, our tracking software package is freely available and provides a simple interface that includes tools for rapid data collection and analysis. By contrast with other tools, it is not dependent on a specific set of hardware. We propose that the tracker may be used for a broad range of additional worm locomotion applications including genetic and chemical screening.