The application of chromosome banding and the new molecular cytogenetics techniques, in particular fluorescent in situ hybridization (FISH) using chromosome painting probes found early use for understanding the systematic and phylogenetic relationships of species, as well as for providing insights to the possible mechanisms underpinning speciation. Here, we showed an application of primed in situ DNA synthesis (PRINS) and fluorescent in situ hybridization for comparative detection of the domestic horse (Equus caballus) and the domestic donkey (Equus asinus) telomere, centromere and nucleolar-organizer region sequences. The number, distribution and kind of highly repeated DNA sequences in across species with regard to their advantage and limit usefulness in phylogenetic analyses are discussed.