In riverine systems, the construction of locks and dams may present barriers to movement for aquatic organisms and effectively fragment otherwise continuous populations. We examined the spatial ecology and population genetics of northern map turtles (Graptemys geographica) in the Trent-Severn Waterway (TSW), Ontario, Canada. We hypothesized that the locks and dams on the TSW are fragmenting map turtle habitats and creating barriers to movement, and predicted that turtles from fragmented habitats have significantly reduced home range sizes and daily movements compared to turtles from unfragmented habitats, and that populations on either side of a lock and dam would be genetically distinct. Home ranges and average daily movements were smaller for turtles from fragmented habitats compared with continuous habitats, indicating that locks and dams are restricting mobility. However, populations in fragmented habitats showed none of the predicted genetic consequences of fragmentation (e.g., reduced heterozygosity and allelic richness, differentiation across a barrier). Genetic data may be reflecting historic population structure because only a few turtle generations have passed since the construction of the locks and dams about 100 years ago. Additionally, our genetic data may not be sufficiently robust to detect differentiation at this relatively small geographic scale. Our study highlights the conservation importance of examining multiple facets of a single potential threat, especially for long-lived species that may show differences in short- and long-term effects of fragmentation.