DNA-based identification of organisms is an important tool in biosecurity, ecological monitoring and wildlife forensics. Current methods usually involve post-polymerase chain reaction (PCR) manipulations (e.g. restriction digest, gel electrophoresis), which add to the expense and time required for processing samples, and may introduce error. We developed a method of species identification that uses species-specific primers and melt-curve analysis, and avoids post-PCR manipulation of samples. The method was highly accurate when trialled on DNA from six large carnivore species from Tasmania, Australia. Because of its flexibility and cost-effectiveness, this method should find wide use in many areas of applied biological science.