In this study, we focused on species identification in leather. Based on optimized extraction method combined with a panel of ultra-specific primers targeting short mitochondrial DNA fragments, we have developed a complete and efficient method that allows molecular identification of species origin in leather. Despite great advances in the fields of forensic, species identification in processed animal products is still very limited, not only because of classic problems as the tiny amount of DNA and/or DNA degradation, but also because of the critical problem of PCR inhibitors which are co-extracted with DNA molecules. In perspective, this study could be applied to detect frauds in particular in leather market and/or protect endangered species.