In the last few years, DNA barcoding became an established method for species identification in biodiversity inventories and monitoring studies. Such studies depend on the access to a comprehensive reference data base, covering all relevant taxa. Here we present a comprehensive DNA barcode inventory of all amphibian and reptile species native to Austria, except for the putatively extinct Vipera ursinii rakosiensis and Lissotriton helveticus, which has been only recently reported for the very western edge of Austria. A total of 194 DNA barcodes were generated in the framework of the Austrian Barcode of Life (ABOL) initiative. Species identification via DNA barcodes was successful for most species, except for the hybridogenetic species complex of water frogs (Pelophylax spp.) and the crested newts (Triturus spp.), in areas of sympatry. However, DNA barcoding also proved powerful in detecting deep conspecific lineages, e.g. within Natrix natrix or the wall lizard (Podarcis muralis), resulting in more than one Barcode Index Number (BIN) per species. Moreover, DNA barcodes revealed the presence of Natrix helvetica, which has been elevated to species level only recently, and genetic signatures of the Italian water frog Pelophylax bergeri in Western Austria for the first time. Comparison to previously published DNA barcoding data of European amphibians and reptiles corroborated the results of the Austrian data but also revealed certain peculiarities, underlining the particular strengths and in the case of the genus Pelophylax also the limitations of DNA barcoding. Consequently, DNA barcoding is not only powerful for species identification of all life stages of most Austrian amphibian and reptile species, but also for the detection of new species, the monitoring of gene flow or the presence of alien populations and/or species. Thus, DNA barcoding and the data generated in this study may serve both scientific and national or even transnational conservation purposes.