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The creation and use of a globally available database of DNA sequences from a standardized gene region has been proposed as a tool for species identification, assessing genetic diversity and monitoring the legal and illegal trade in wildlife species. Here, we contribute to the Barcode of Life Data System and test whether a short region of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene would reliably distinguish among a suite of commonly hunted African and South American mammal and reptile species. We used universal primers to generate reference barcode sequences of 645 bp for 23 species from five vertebrate families (Crocodilidae, Alligatoridae, Bovidae, Suidae and Cercopithecidae). Primer cocktails yielded high quality barcode sequences for 179 out of 204 samples (87.7%) from all species included in the study. For most taxa, we sequenced multiple individuals to estimate intraspecific sequence variability and document fixed diagnostic characters for species identification. Polymorphism in the COX1 fragment was generally low (mean = 0.24%), while differences between congeneric species averaged 9.77%. Both fixed character differences and tree-based maximum likelihood distance methods unambiguously identified unknown and misidentified samples with a high degree of certainty. Barcode sequences also differentiated among newly identified lineages of African crocodiles and identified unusually high levels of genetic diversity in one species of African duiker. DNA barcoding offers promise as an effective tool for monitoring poaching and commercial trade in endangered species, especially when investigating semi-processed or morphologically indistinguishable wildlife products. We discuss additional benefits of barcoding to ecology and conservation.