The herpetofauna of São Tomé and Príncipe consists of nine species of amphibians, all endemic, and 21 species of terrestrial reptiles, of which 17 are endemic. Our current knowledge regarding its natural history, ecology, and distribution is limited. Here two important tools are provided to support researchers, conservationists, and local authorities in the identification of the country’s herpetofauna: an illustrated key to the herpetofauna of the two islands and surroundings islets and a DNA barcode reference library. The keys allow a rapid and unambiguous morphological identification of all occurring species. The DNA barcodes for the entire herpetofauna of the country were produced from 79 specimens, all of which are deposited in museum collections. The barcodes generated are available in online repositories and can be used to provide unambiguous molecular identification of most of the species. Future applications and use of these tools are briefly discussed.