Global climate models predict increases in mean temperature and changes in thermal variability. Despite predicted changes in temperature variability and evidence of its severe effects on ecological communities, research has mainly focused on changes in mean temperatures. We test fundamental ecological theories regarding how seasonal and daily temperature variability affect the temperature tolerance of aquatic macroinvertebrates, and physiologically constrain them in the landscape. Predicting organismal response to climate change requires an understanding of the mechanisms by which temperature variability impacts species’ performance at large spatial scales. We compare critical thermal limits of aquatic insects from the orders Ephemeroptera (mayflies), Plecoptera (stoneflies), and Trichoptera (caddisflies) in temperate and tropical montane Australia with existing data from the Americas. Our study design incorporates an intercontinental comparison between temperate locations (high seasonal variability) and tropical locations (low seasonal variability) across an elevation (thermal) gradient. Across all insect orders, our results show wider thermal tolerance breadth in the temperate locations relative to the tropical locations in agreement with Janzen’s Climate Variability Hypothesis. Our results also show that Plecoptera generally had wider thermal tolerance breadth than Ephemeroptera and Trichoptera in contrast to the usual assumption that Plecoptera are generally cold-water specialists. Overall our findings suggest that tropical stream insects, especially species of Ephemeroptera and Trichoptera, may be more vulnerable to acute thermal stress from climate change than temperate species. An improved understanding of how organisms will respond to future thermal regimes can be achieved through further comparisons of organisms’ thermal tolerances of organisms from locations which vary in terms of mean and variability of temperature.