We report the development of a fast and reliable PCR-based method for sex identification of African Rhinoceros (Ceratotherium simum and Diceros bicornis) that could easily be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. In both species, the amplified ZFX and ZFY amplicons differ in size by 7 bp and can thus be differentiated by capillary electrophoresis. Blood, tissue, horn, and faecal samples were correctly sexed using this method. Cross species testing also demonstrated that this method could be used to sex Indian rhinoceros (Rhinoceros unicornis) samples.