The European red fox (Vulpes vulpes) is a highly adaptable predator indigenous to the northern hemisphere. However, in Australia the red fox is a widespread exotic predator that has contributed to the decline and extinction of several native species. Here we describe a multiplex PCR assay for the molecular identification of the red fox. The identification is achieved by the generation of a diagnostic profile combining the lengths of mitochondrial ribosomal RNA (rRNA) gene regions amplified using highly conserved PCR primers. The method was tested in DNA samples from 17 species, including in mixtures. Our results demonstrate that the red fox has a unique combination of fragment lengths determined by capillary electrophoresis that can be used for its unambiguous discrimination from common domestic and wild species.