DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present a part of the InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of cuckoo wasps (Hymenoptera, Chrysididae) dataset representing 144 specimens and 103 species, covering approximately 44% of the Iberian and 21% of the European fauna. The InBIO Barcoding Initiative (IBI - DNA Barcoding Portuguese terrestrial invertebrate biodiversity) aims to fill the barcoding gap for the terrestrial invertebrate taxa. All DNA extractions are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and specimens are deposited in the University of Mons collection (Belgium) and in the Natur-Museum in Lucerne (Switzerland). This dataset increases the knowledge on the DNA barcodes and distribution of 102 species of cuckoo wasps. A total of 52 species, from 11 different genera, were new additions to the Barcode of Life Data System (BOLD), with DNA barcodes for another 44 species added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through the BOLD online database. Nine cuckoo wasp species are newly recorded for Portugal. Additionally, two new species for science are described: Chrysiscrossi Rosa, sp. nov. from southern Portugal and Hedychridiumcalcarium Rosa, sp. nov. from eastern Spain. Several taxonomic changes are proposed and Hedychrumrutilans Dahlbom, 1845 is found to consist of two different taxa that can be found in sympatry, Hedychrumrutilans s. str. and Hedychrumviridaureum Tournier, 1877 stat. nov. Stilbumwestermanni Dahlbom, 1845 stat. nov. is confirmed as distinct from Stilbumcalens (Fabricius, 1781), with the latter species not confirmed as present in Iberia; barcoded Stilbum material from Australia is distinct and represents Stilbumamethystium (Fabricius, 1775) sp. resurr.; Portuguese material identified as Hedychridiumchloropygum Buysson, 1888 actually belongs to Hedychridiumcaputaureum Trautmann & Trautmann, 1919, the first confirmed record of this species from Iberia. Philoctetesparvulus (Dahlbom, 1845) is confirmed to be a synonym of Philoctetespunctulatus (Dahlbom, 1845). Chrysislusitanica Bischoff, 1910 is confirmed as a valid species. Chrysishebraeica Linsenmaier, 1959 stat. nov. is raised to species status.