Flow cytogenetics is a high-throughput technique that classifies large populations of mitotic chromosomes according to their fluorescence and light scattering as they move in a single file rapidly in a narrow stream of liquid. Flow karyotyping can detect structural and numerical chromosome changes, and chromosomes purified by sorting have been used to discover the three-dimensional organization of DNA, characterize the proteome, and describe the organization of the perichromosomal layer at the nanometre level. However, the most frequent and important use of flow cytogenetics has been the isolation of different chromosomes. Chromosome sorting has made it possible to dissect nuclear genomes into small and defined parts, therefore allowing targeted, simplified, and more economical genomic analyses. The uses of flow-sorted chromosomes for genome complexity reduction and targeted analysis have expanded hand-in-hand with the progress of molecular biology and genomics techniques. These uses include the targeted development of DNA markers, the construction of chromosome-specific DNA libraries, physical mapping, draft genome sequencing, and gene cloning. Graphical abstract: [Figure not available: see fulltext.]. © 2023, The Author(s).

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