The potential to migrate is one of the most fundamental functions for various epithelial, mesenchymal, and immune cells. Image analysis of motile cell populations, both primary and cultured, typically reveals an intercellular variability in migration speeds. However, cell migration chromatography, the sorting of large populations of cells based on their migratory characteristics, cannot be easily performed. The lack of such methods has hindered our understanding of the direct correlation between the capacity to migrate and other cellular properties. Here, we report two novel, easily implementable and readily scalable methods to sort millions of live migratory cancer and immune cells based on their spontaneous migration in two-dimensional and three-dimensional microenvironments, respectively. Correlative downstream transcriptomic, molecular and functional tests reveal marked differences between the fast and slow subpopulations in patient-derived cancer cells. We further employ our method to reveal that sorting the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This phenotypic assay opens new avenues for the precise characterization of the mechanisms underlying hither to unexplained heterogeneities in migratory phenotypes within a cell population, and for the targeted enrichment of the most potent migratory leukocytes in immunotherapies.