For several groups of insects, including the velvet ants, male–female associations are difficult because of extreme dimorphism between the sexes. Many species, or even genera, are known from only a single sex. In this study, we investigate the use of DNA sequences of the internal transcribed spacer regions (ITS1 & ITS2) as a means of associating males and females in velvet ants. We compare ITS sequences between sexes in taxa where the associations are well documented and uncontested. We compare these sequences to other closely related species. The DNA sequence data show little or no variability between sexes, while much greater differences exist between species. This method should prove a valuable technique for making associations between previously unmatched males and females in velvet ants, and likely other hymenopteran taxa.