Freshwater catchments of south-east Australia possess generally rich and diverse macroinvertebrate faunas, although the genetic structuring of these assemblages is poorly known. In this study, we assessed mitochondrial phylogenetic structure within four genera of macroinvertebrates from the Sydney Water Supply Catchment, south-east Australia: Euastacus (parastacid crayfish), Cheumatopsyche (hydropsychid caddisflies), Atalophlebia (leptophlebiid mayflies) and Paratya (atyid shrimp), with a view to prioritising areas of high diversity for future conservation efforts. We found extremely divergent (≈4–19%) cytochrome c oxidase subunit I (COI) lineages within all surveyed groups, many of which corresponded to recognised taxa, although there was also evidence of cryptic species within three genera; Euastacus, Atalophlebia and Paratya. Distributions of these three genera were associated with high altitude streams, above dam impoundments. Our results have important implications for management of the Sydney Water Supply Catchment. Future disturbance in this region is likely to be high and priority should be directed towards preserving the diversity of fauna in these upland areas. This comparative phylogenetic approach may have value as a means to focus and direct conservation efforts in other areas supporting high biodiversity.