As global freshwater biodiversity continues to deteriorate rapidly, it is critical that system-specific conservation actions are identified and implemented. In interconnected freshwater systems like waterways and canals, foremost threats include river fragmentation and connectivity loss, and the diffusion of invasive species. However, managing barrier (e.g., locks, dams) connectivity in waterways to selectively allow and restrict passage of native and invasive species, respectively, is a major global challenge. Canada's historic Rideau Canal Waterway, located in eastern Ontario, is interconnected by 24 operating lockstations and embodies this challenge. To minimize invasive species dispersal, without compromising connectivity to native species, we must first assess if, when, and to what extent native and invasive fishes move throughout the waterway and the potential factors influencing movements.Within each reach (i.e., the waters between two lockstations), connectivity may also seasonally vary due to ice formations and reduced flows and/or water levels, though literature on this topic is currently limited. Here, we blended several fish tracking methods with ecohydraulics and management consultations to evaluate system connectivity and fish movement patterns. Three native fishes, largemouth bass (Micropterus salmoides; N = 56), muskellunge (Esox masquinongy; N = 23), and northern pike (Esox lucius; N = 114), and two invasive fishes, common carp (Cyprinus carpio; N = 54) and round goby (Neogobius melanostomus; N = 45), were acoustically tagged and tracked in 2019-2021 across 45 km of the waterway. Additionally, we externally (anchor) tagged 9564 individuals across 15 species, with tags deployed in 2018-2023, to evaluate potential passages and general space use.Movement data suggests that lockstations reduce, but not entirely restrict, connectivity, and that seasonal water-level drawdowns further reduce connectivity within river reaches. We found that most passages were in the direction of flow, a potential issue to native upstream migratory species. Notably, while we documented several native fish species conducting inter-reach movements, no common carp passages were detected and only a single round goby moved upstream through a lock. Results from this research add to the limited, but growing, literature base of native and invasive fish movements in waterways, providing evidence and suggestions for managers and conservation groups to develop selective fish passage strategies or take restorative actions. Given that waterways are pervasive globally, this research informs not only the conservation and management of fish populations in the Rideau Canal Waterway, but of other waterways in Canada and beyond. Chapter 4: Multi-year evaluation of muskellunge (Esox masquinongy) spatial ecology during winter drawdowns in a regulated, urban waterway in Canada……………………….………………87 4.1. Abstract………………………………………………………….…….……………………………………87 4.2. Introduction…………………………………………………..………….…………………………………87 9 4.3. Materials and methods……………………...