Freshwater crayfish play important ecological roles as ecosystem engineers, detritivores, and prey for a range of aquatic and terrestrial predators. Despite their importance in river ecosystems, little is known about how they respond to river regulation. Dams alter natural flow regimes and can profoundly affect aquatic ecosystems, yet the consequences for Australian spiny crayfish remain largely unknown. We examined the effects of five large dams on populations of the Giant Spiny Crayfish (Euastacus spinifer), one of the world's largest freshwater invertebrates, to determine how flow regulation influences population structure and abundance. Using a reference–impact study design, we surveyed eight regulated and seven unregulated sites within the Sydney drinking water catchment. Sites were sampled on two occasions over a two-week period using baited traps and baited remote underwater videos. We measured abundance, catch per unit effort, occipital carapace length, weight, condition factor and ectocommensal loads to assess the effects of river regulation on crayfish populations while accounting for other environmental factors. River regulation had a strong negative effect on crayfish abundance, with regulated sites supporting significantly fewer Euastacus spinifer than unregulated reference sites. We detected no clear differences in condition, weight or carapace length; however, these population metrics could not be robustly assessed because of the extremely low abundance of crayfish at regulated sites. These findings provide quantitative evidence that large dams may substantially affect populations of Australian spiny crayfish and highlight the need to consider crayfish in environmental flow management. Given the ecological importance and conservation significance of the genus Euastacus, the potential effects of river regulation and flow alteration on other species warrant further investigation.