FISH using whole-chromosome or region-specific painting probes, derived from either flow-sorted or microdissected chromosomes, has revolutionized cytogenetics. The generation of paints/ painting probes from flow-sorted chromosomes requires both an expensive, sophisticated fluorescence-activated cell sorter and suspensions of freshly prepared chromosomes. In contrast, producing paints from microdissected material requires an inverted microscope equipped with micromanipulators and metaphase chromosome spreads on coverslips. Painting probes generated from flow-sorted chromosomes typically provide better chromosomal coverage and can be applied across distantly related species, whereas region-specific probes from microdissection offer higher resolution, but are generally limited to comparative painting between closely related species. Here, we present detailed protocols for the generating probes from both flow-sorted and microdissected chromosomes.