Current species profiling techniques usually require several steps to identify an unknown species in quarantine cases and other forensic applications. Here we have developed a species profiling test that produces unique profiles for all vertebrate species tested using a single primer in a polymerase chain reaction. Samples tested included a range of mammals and other vertebrates such as fish and marsupials; a group of animals yet to be characterized with molecular speciation techniques. Species-specific profiles were shown to be reproducible and able to be generated from less than 10 ng of total DNA, comparable to DNA quantities used in conventional species profiling techniques. A case study demonstrates the utility of the technique by distinguishing between commercial and protected species of the Macropodidae (kangaroo) superfamily.