Citation: Brewitt, K. S., and E. M. Danner. 2014. Spatio-temporal temperature variation influences juvenile steelhead (Oncorhynchus mykiss) use of thermal refuges. Ecosphere 5(7):92. http://dx.doi.org/10.1890/ES14-00036.1Abstract. Thermal refuges form potentially critical habitat for species at the limits of their thermal tolerance, especially given large-scale habitat degradation and rising temperatures across ecosystems. The Klamath River is a highly altered system where summer mainstem temperatures reach levels that are physiologically stressful to threatened Pacific salmonid populations, making thermal refuges critical for over-summer survival when temperatures near upper thermal thresholds. Small changes in water temperature can have a large effect on salmonid growth and survival, and therefore fine-scale spatiotemporal temperature variation could influence when and where refuges are important for both individual survival and population persistence. In this study, we combined monitoring of environmental variables with measures of fish temperature (a proxy for refuge use) to quantify juvenile steelhead (Oncorhynchus mykiss) use of thermal refuges. We used a logistic mixed effects model to determine the relative influence of instantaneous mainstem temperature and flow, sub-daily temperature variation, body size, and time of day on steelhead refuge use. Mainstem temperature was the strongest predictor of refuge use; the majority (.80%) of juvenile steelhead moved into refuges when mainstem temperatures reached 22-238C, and all fish moved in by 258C. Fish were more likely to use refuges with increased diel mainstem temperature variation and larger temperature differential between the mainstem and tributary. In addition, steelhead exhibited a distinct diel behavioral shift in refuge use that varied with body size; smaller juveniles (;160 mm) were much more likely to use refuges during the night than day, whereas larger juveniles (;210 mm) exhibited a much less pronounced diel behavioral shift. Given impacts of watershed alteration and climate change and the growing importance of refuge habitat, these findings suggest that species persistence may depend on extremely fine-scale spatial and temporal temperature dynamics.