Using the random amplified polymorphic DNA (RAPD) technique, a fragment (OPA13₅₉₀) was developed from Oreochromis niloticus. The fragment was cloned and single-locus-specific probes were designed using the sequence-characterized amplified region (SCAR) technique. The probes were used on DNA from different species of tilapia (O. niloticus, O. aureus, O. mossambicus, O. spilurus and Tilapia mariae). The amplification products of the RAPD marker with lengthened specific primers (SCAR-RAPD) revealed length variation between species, but not within species. Mendelian inheritance of the SCAR-RAPD marker was shown in 32 backcrosses from matings between O. niloticus and O. aureus.