Population structure is influenced by extrinsic factors, such as landscape architecture and dispersal barriers. Lotic network architecture is known to constrain ecological, demographic and evolutionary processes, including population genetic structure. I assessed the population structure of a widespread aquatic salamander, Necturus maculosus, across three river basins in central and eastern Kentucky. I examined the role of network architecture, anthropogenic barriers, and spatial scale on patterns of population structure. I also provided a review of N. maculosus capture methods and offer an improved trap design. I identified significant structuring between the combined Licking/Kinniconick basin and the Kentucky River basin, with further structure within each basin. I found evidence for both hierarchically organized populations structure (e.g. Stream Hierarchy Model), as well as population structure unaffected by network hierarchy (e.g. Death Valley Model). These results highlight the importance of scale when examining population structure. Whereas one model may suffice to explain population structure at a local scale, a second model may be necessary to accurately describe the population structure across larger spatial scales. These results suggest that local factors affect population structure uniquely across a species’ range, and support a multi-model approach for assessing population structure.