The identification of causal genomic loci and their interactions underlying various traits in plants has been greatly aided by progress in understanding the organization of the nuclear genome. Flow cytometric sorting of condensed mitotic chromosomes makes possible to purify single chromosomes in large quantities, and as the DNA remains intact this process can be coupled successfully with many techniques in molecular biology and genomics. Since the first experiments demonstrated the possibility of isolating particular chromosomes by flow cytometric sorting, numerous uses of flow-sorted chromosomes in molecular biology, genomics and proteomics have been developed. These applications were facilitated by the ability to purify chromosomes in quantities required for particular applications and made the sorted chromosomes an invaluable resource for important endeavors, including large international projects on sequencing complex plant genomes. This review discusses these applications, describes their contribution to advancements in plant genome analysis and gene cloning, and outlines future directions.