We described a technique for detecting mammal species, based on the analysis of a control region fragment of mitochondrial DNA by establishing taxonomic identity from non-invasive samples. We detected a polymorphic fragment that varies in sequence and length within different mammalian species but maintains its identity among individuals of the same species. We ampliﬁed a single fragment in all the mammalian species tested from tissue samples and identiﬁed feces samples at species level. The use of a unique set of primers to assess the presence of different mammal species with non-invasive sampling allowed us to differentiate sequences from more than one species per environmental sample. Thus, it constitutes a powerful molecular tool for inventory and description of the mammal diversity distribution in natural areas.