Freshwater organisms are threatened by changes in streamflow and water temperature regimes due to global climate change and anthropogenic activities. Threats include the disappearance of narrow-tolerance species and loss of favorable thermal conditions for cold-adapted organisms. Mayflies are an abundant and diverse indicator of river health that performs important functional roles. The relative importance of key hydro-environmental factors such as water temperature and flow volumes in structuring these communities has rarely been explored in the tropical regions of Africa. Here, we investigate the response of mayfly species diversity to these factors in the Luvuvhu catchment, a strategic water source area in the arid northeastern region of South Africa. Mayfly larvae were sampled monthly in stones-in-current biotopes across 23 sites over a one-year period. The relationship between these environmental drivers and mayfly diversity was modeled using linear mixed-effects models (LMMs) and a model-based multivariate approach. Threshold Indicator Taxa Analysis (TITAN) was used to model the response of mayfly species to important gradients and identify thresholds of change. Site-specific characteristics were the most important predictor of mayfly diversity, and there was considerable variation over time, with mayfly diversity peaking during winter. Along with this, gradient temperature was the best predictor of assemblage structure, with five out of six reliable indicator species being cold-adapted, and a community threshold response at 19°C. Results support laboratory-based thresholds of temperature for mayfly species survival and development, extending empirical evidence to include field-based observations. Increased global (climate change) and local (riparian vegetation removal, impoundments) changes are predicted to have negative impacts on mayfly diversity and ultimately on ecosystem function.