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Blackflies are amongst the most abundant and diverse group of aquatic insects globally, being rarely absent from rivers, their distributions are largely driven by current velocity, conductivity and total dissolved solids (TDS). While research on blackflies has mainly focused on medical, veterinary and economic aspects, ecological studies in the Afrotropical Region are relatively scarce. This study aimed to determine the response of blackfly species to environmental drivers in the north-eastern tropical regions of South Africa. Generalized Linear mixed models and the Permutational Multivariate Analysis of Variance were fitted to model blackfly richness and abundance structure. The Threshold Indicator Taxa Analysis (TITAN) was used to analyze species-specific change along environmental gradients. A total of 1343 larvae, representing seven blackfly species in four sub-genera, were collected from 22 sites over a twelve-month period. Water temperature and flow velocity were the most important drivers of species richness. Conductivity and land cover had the strongest effect on community composition across species and sites. Species responded synchronously along flow velocity and water temperature gradients at 1.6 m.s−1 and between 20–22 °C, respectively. Simulium (Metomphalus) hargreavesi was identified at an indicator species for flow velocity, while three were indicator species for water temperature change. We demonstrated that the richness and relative abundance of blackflies are driven by different variables across the Luvuvhu catchment. The new information obtained from this study provides an insight on the importance of protecting and managing the natural environment in a high biodiversity and endemism area. [ABSTRACT FROM AUTHOR]