Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique used to physically map DNA sequences on chromosomes. In FISH, DNA probes are most often hybridized to target DNA in metaphase chromosomes spread on slides (in situ). FISH probes are variable in size. The largest FISH probes are chromosome “paints,” which are made by flow sorting, followed by PCR amplification. Chromosome paints are specific for individual, whole chromosomes. Chromosome paints have been used to map the chromosome homology between humans and many primate species. Smaller cloned DNA probes, for example bacterial artificial chromosomes (BACs), can reveal intrachromosomal rearrangements such as inversions. Comparisons between primate species using BAC–FISH have been used to identify differences in marker order between primate species. A cladistic analysis helped establish the polarity of changes in marker order. One outcome was the discovery that centromere shifts or evolutionary new centromeres (ENC) have been a common feature of chromosome evolution in primates.