When riverine landscapes become fragmented by human impacts such as dams, constraints to gene flow can drive increased isolation between populations. The effects that dams have on connectivity among aquatic insect populations is largely understudied and, for most species, based on assumptions about dispersal ability. We used Next-generation sequencing to generate mitochondrial COI markers to study the spatial genetic structures of two caddisflies, Triplectides ciuskus and Notalina bifaria, and a stonefly, Leptoperla primitiva, among sites that were either separated or not-separated by a large dam in north-eastern Victoria, Australia. Despite a strong flight ability, population connectivity in T. ciuskus was significantly affected by the presence of Dartmouth Dam. For the other two species, the dam had no apparent effect. Results showed that connectivity of populations could not reliably be inferred from the assumed dispersal abilities of the species, with the species considered to be a strong flier (T. ciuskus) showing significant population fragmentation, while another species considered to have poor dispersal ability (L. primitiva) showed no fragmentation. Thus, our results suggest that, in order to understand the effects of dam-led fragmentation on aquatic species, population structure of individual species needs to be investigated rather than assumed through dispersal traits.