Identification of macroinvertebrates is a key component of monitoring programs that seek to understand the condition of aquatic environments. Classical identification approaches underpin such programs, but molecular approaches are gaining recognition as valuable ways to identify organisms for research and monitoring programs. We applied DNA barcoding data to specimens collected as part of monitoring programs in the Murray–Darling Basin, to investigate the possible informational benefits these data may provide. We also tested the performances of two online DNA databases in assigning taxon names to our sequence data. We found that relying on the online databases to determine species identifications was currently problematic for the Australian freshwater fauna because of a lack of available sequence data. However, we also found that collecting and applying barcode data to our monitoring programs gave considerable informational benefits by providing greater resolution of specimen identity, highlighting the presence of potential cryptic species, providing information on larval and adult associations, demonstrating instances where misidentification had occurred though classical approaches, and providing conformation of the performance of diagnostic characters currently used in keys to determine species identities.