FISH with whole-chromosome or region-specific painting probes made from either flow-sorted or microdissected chromosomes has revolutionized cytogenetics. The generation of paints from flow-sorted chromosomes relies on the use of an expensive and sophisticated fluorescence-activated cell sorter and suspensions of freshly prepared chromosomes. The preparation of paints from microdissected materials requires an inverted microscope with appropriate micromanipulators and metaphase chromosome spreads on coverslips. Painting probes made from flow-sorted chromosomes generally have better chromosomal coverage and can be used in a wide range of applications between unrelated species, while region-specific probes from microdissection enable higher resolution analyses restricted to comparative painting between closely related species. Here we provide detailed protocols on the generation of probes from both flow-sorted and microdissected chromosomes.