Studies on feeding habits based on faeces dissecting are imperative to understand the natural history of Tibetan foxes and their functions in the transmission of a lethal zoonotic parasite, Echinococcus multilocularis. However, Tibetan foxes and red foxes live sympatrically on the Tibetan plateau, China. Therefore, the faeces of Tibetan foxes must be distinguished from those of red foxes. We established a diagnostic method to distinguish the faeces of the two species by amplifying a portion of the mitochondrial cytochrome b gene (cytb) and digesting with the restriction enzymes BamHI and SspI, to produce specific diagnostic banding patterns. This PCR-RFLP assay enabled rapid, accurate and easily performed identification and differentiation of the two species.