As global populations grow, demand for generation of affordable and efficient electricity will likely increase, requiring tradeoffs between power generation and ecosystems sustainability, including water quality and species habitat. Once-through thermoelectric power plants, representing 30% of the electricity generation in the United States, withdraw and discharge large quantities of water for cooling purposes. This process can cause thermal pollution in waterways, adversely affecting aquatic communities. Incorporating biology into the energy-water nexus can aid decision-makers in identifying tradeoffs and more effectively assessing and managing aquatic ecosystems. To quantify thermal pollution and the risk posed to aquatic species, we created an adaptable, novel methodology that utilizes plume mixing and probability distribution analyses on temperature and flow data for both a power plant's discharge and the adjoining river. To assess risk, we developed a probability risk space that quantifies the probability of exceeding a given temperature. The Shawnee Fossil Plant on the Ohio River was selected to demonstrate the methodology, and three fish species with associated upper thermal avoidance limits were selected for comparison. Our results highlight that both the lateral and longitudinal location from the point of effluent mixing within the river affects the probability of thermal risk to aquatic species. A high degree of risk within a plume can reduce to a smaller total risk within the context of a large river cross-section. Our results emphasize the need for individualized risk assessment for Clean Water Act 316(a) requirements for power plant effluent temperature limits and National Pollutant Discharge Elimination System permits. These findings are applicable in policy-making, environmental mitigation, and power plant operations management.