Hyporheic zones contribute to lower temperatures in many rivers, creating a longitudinal heterogeneous array of thermal refuges. In this study, we had the unique opportunity to show temperature reduction along actual hyporheic zone pathlines in a large river system that contribute to the maintenance of refuges through discharge into off-channel habitats. Temperature was monitored in a dense network of wells that were located along pathlines in small islands, from a calibrated ground-water flow model. Temperature along one 600-m pathline was reduced about 7 °C. Among three islands that were adjacent to the river, the northern two showed exponential decrease in temperature with distance, with fitted thermal Péclet numbers of 2.7 and 6.5, while the southern island showed no significant decrease. We suggest this is due to the higher infiltration rate in the wet season in this larger, more mature island, which suppresses hyporheic flow in the wet season. Stable isotope sampling showed that values of δ2H were higher in areas where we observed lower temperatures. The overall relationship of δ2H versus temperature was significant with a slope of -0.329. This implies that lower temperatures are associated with water that has had contact with deeper groundwater or that lower temperatures have been affected by local rainfall infiltration, or water that has entered the hyporheic zone in winter. These findings are important because they allow estimation of the temperature benefit that may be achieved in similar geomorphic settings, providing implications for riparian restoration.