A recent DNA barcoding study of Australian microgastrines (Hymenoptera: Braconidae) sought to use next generation sequencing of the cytochrome c oxidase subunit 1 (COI) barcoding gene region, the wingless (WG) gene and the internal transcribed spacer 2 (ITS2) to delimit molecular species in a highly diverse group of parasitic wasps. Large intragenomic distances between ITS2 variants, often larger than the average interspecific variation, caused difficulties in using ITS2 for species delimitation in both threshold and tree‐based approaches, and the gene was not included in the reported results of the previous DNA barcoding study. We here report on the intragenomic, and the intra‐ and interspecies, variation in ITS2 in the microgastrine genus Diolcogaster to further investigate the value of ITS2 as a marker for species delimitation and phylogenetics of the Microgastrinae. Distinctive intragenomic variant patterns were found in different species of Diolcogaster, with some species possessing a single major variant, and others possessing many divergent variants. Characterising intragenomic variation of ITS2 is critical as it is a widely used marker in hymenopteran phylogenetics and species delimitation, and large intragenomic distances such as those found in this study may obscure phylogenetic signal.