Hydrokinetic energy resource assessment is a crucial prerequisite for strategic turbine deployment and energy extraction. Despite advancements in analytical tools, resource assessment is often completed without detailed investigation of spatial and temporal flow variation and implications on optimal turbine placement. A case study was conducted on the Rivière Rouge, Québec, Canada to estimate the hydrokinetic energy resource, to locate the optimal turbine placement, and to study the impact of seasonal flow variation. The primary optimal turbine location did not change, but the second, third, and fourth optimal locations were impacted. Assuming a hypothetical deployment of one turbine with a 1 m2 swept area, the theoretical hydrokinetic energy resource for the site was 21.8 MWh per year in the optimal turbine locations and 6.2 MWh per year using the reach-averaged velocity. This difference illustrates the need to consider the entire velocity flow field in hydrokinetic energy assessments. To conduct the assessment, field data were collected with an acoustic Doppler current profiler and a global positioning system for hydrodynamic model generation, calibration, and validation using the software TELEMAC-2D. The mean absolute percentage errors of the model in the areas of interest were 14.8% for calibration and 22.9% and 19.4% for validation.