Comparative genomic hybridization (CGH) is a refined molecular cytogenetic method based on the standard fluorescence in situ hybridization protocol but using whole genomes as probes instead of short, defined sequences. Essentially, two or more different genomes labeled with specific fluorochromes or haptens are hybridized against the chromosomal background of selected target species, often using competitive DNA to block common repetitive regions, aiming to reveal specific chromosomal segments (i.e., in sex chromosomes) or parental sub-genomes (in hybrids). This approach, originally designed for clinical diagnostics, has been now effectively employed to examine various evolutionary phenomena in diverse metazoan groups. Here, we delimit the CGH design, describe the protocol along with possible applications and associated method’s limitations, and compile several illustrative examples across both vertebrate and invertebrate animal groups.