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Regulated waterways, interconnected by navigation barriers (locks and dams), are uniquely difficult to manage, given interest in enabling native species connectivity while minimizing invasions. Canada's historic Rideau Canal Waterway, a 202 km navigable route located in eastern Ontario and connected by 24 lockstations, embodies this challenge. The lock(s) and water-control dam that compose each lockstation may respectively offer a connectivity pathway, though to what extent is unclear. We used acoustic telemetry (native largemouth bass (Micropterus nigricans) and northern pike (Esox lucius), and invasive common carp (Cyprinus carpio); n = 224) to evaluate fish connectivity relative to lock operations and environmental data over three years (2019–2021). Thirty-five passages by 23 native fishes were recorded, with 49% of passages through locks. No common carp passages were detected; movements indicate that they favour higher flow areas downstream of dams, regions with no pathway upstream. Most passages were downstream and, of concern to obligate upstream migrators, we found that multi-flight and higher-lift locks appear impassable to upstream movements. Our results suggest that these lockstations limit, but not entirely restrict, connectivity. [ABSTRACT FROM AUTHOR]