Whole chromosome paints for human (HSA) chromosomes 9, 12, 15 and 20 and arm-specific paints for HSA16p, 19p and 19q were applied on donkey metaphase spreads. All probes, except HSA19p, gave distinct hybridization signals on donkey chromosomes/chromosomal segments. The results show direct segmental homology between human and donkey genomes, and enable refinement of correspondence between donkey and horse karyotypes. Of specific interest is the identification of hitherto unknown correspondence between four equine acrocentric chromosomes (ECA22, 23, 25 and 28) and the donkey chromosomes. Overall, the findings mark the beginning of an ordered study of comparative organization of genomes/karyotypes of the equids, that can shed light on karyotype evolution and ancestral chromosomal condition in the Perissodactyls.