Background Identifying and isolating cells with specific behavioral characteristics will facilitate the understanding of the molecular basis regulating these behaviors. Although many approaches exist to characterize cell motility, retrieving cells of specific motility following analysis remains challenging. Results Cells migrating on substrates coated with fluorescent microspheres generate non-fluorescent tracks as they move and ingest the spheres. The area cleared by each cell allows for quantitation of single cell and population motility; because individual cell fluorescence is proportional to motility, cells can be sorted according to their degree of movement. Using this approach, we sorted a glioblastoma cell line into high motility and low motility populations and found stable differences in motility following sorting. Conclusion We describe an approach to identify, sort, and enrich populations of cells possessing specific levels of motility. Unlike existing assays of cell motility, this approach enables recovery of characterized cell populations, and can enable screens to identify factors that might regulate motility differences even within clonal population of cells.