Structural and genetic characterization of chromosomes is necessary to understand both normal and pathologic physiology in any species. Flow cytometry and cell sorting technologies provide a means for precise measurement of chromosomal makeup as well as for the isolation of specific chromosomes for further study. Advancements in molecular biology protocols and pressures from large-scale sequencing endeavors placed increased demand on the developers of these instruments for enhanced throughput and quality of results. The ensuing improvements in sorting performance led to the development of a new generation of cytometers known as high-speed cell sorters. These machines provide superior results in less time and are cheaper and simpler to operate than their predecessors. Robust chromosome sorting can now be performed in the laboratories of individual investigators for a variety of gene- and sequence-specific studies. Resolution of the flow karyotype with increased refinement, and the development of new applications for this technology will assure that cell sorting continues to play an important role in cytogenetics, our understanding of molecular processes such as evolution and disease etiology, and ultimately serve as a launching point for predictive medicine.