Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.