The invention of cross-species chromosome painting (Zoo-FISH) represents the most significant technical breakthrough in animal cytogenetics after the introduction of chromosomal banding techniques in late 1960 and early 1970s. This made it possible to compare the karyotypes of virtually any two vertebrate species that diverged up to 100 million years ago. With the availability of paint probes for more and more vertebrate species, Zoo-FISH has made a far-reaching impact on animal comparative cytogenetics, leading to the birth of the new cytogenetics—cytogenomics. Here we present two detailed protocols for cross-species chromosome painting.