With 450 wild bee species, Vienna is among the most species-rich cities of Central Europe. Its geographic position at the intersection of the Atlantic and Continental climate, surrounded by spacious natural areas like the Wienerwald, the Lobau or the Lainzer Tiergarten, combined with the structure-rich microclimate in the city, Vienna is a biodiversity hotspot for wild bees. The aim of the project was to establish DNA-barcoding references from wild bees collected in Vienna and to provide a basis for further molecular studies. A total of 130 wild bee species from 22 genera were analysed and COI sequences were obtained. In addition to the traditional Sanger-sequencing method, which is the standard method for DNA-barcoding, the next-generation sequencing method Illumina MiSeq was successfully applied. Sequences derived by Illumina were shorter with 418 base-pairs in comparison to sequences generated with Sanger with 650 base-pairs. By overcoming some general limitations using Sanger-sequencing, species assignment was effective using the Illumina midi-barcodes and provided other biologically relevant information. The project provided DNA-barcodes of a third of all known wild bees in Vienna and demonstrated the potential of new molecular approaches.