A molecular cytogenetic approach that facilitates high-resolution comparative mapping of defined human genes in different primate species is presented. Fluorescence in situ hybridization and digital imaging microscopy were used to visualize human DNA probes on simultaneously banded or "painted" metaphase chromosomes of great apes (Pan troglodytes, Gorilla gorilla, Pongo pygmaeus), hylobatids (Hylobates lar and Hylobates syndactylus), and Old World monkeys (Macaca fuscata and Cercopithecus aethiops). Using a series of DNA probes, chromosomal rearrangements in the karyotypes of primates were readily detected at the molecular cytogenetic level. This approach should contribute considerably to the understanding of primate phylogeny and evolution.