Freshwater ecosystems are increasingly threatened by landscape-scale environmental changes, making it imperative to understand how these factors influence benthic insect assemblages. Identifying critical thresholds and drivers at the landscape scale is essential to inform effective conservation and management strategies. We evaluated the impact of spatial and climate variables, land use, soils, lithology, and hydrology on benthic insect assemblages across groundwater, runoff, and intermittent flow regimes in the Interior Highlands. Using a biological dataset comprising 210,158 individual records from 609 genera, we applied Gradient Forest analysis to identify environmental thresholds across multiple flow regimes. Spatial and climate variables were the primary drivers for groundwater and intermittent streams while soils and lithology influenced patterns in groundwater and runoff streams. Hydrologic metrics such as frequency, magnitude, rate, and timing varied in importance depending on the flow regime. For intermittent streams, assemblage-level responses were linked to thresholds at lower annual minimum timing values. Groundwater systems exhibited taxa-specific thresholds, whereas runoff streams demonstrated assemblage-level thresholds driven by variability in flow reversals. These findings underscore the critical role of spatial structure, temperature, and lithology across all flow regimes, with hydrology contributing differently based on flow regime and taxa. Environmental thresholds were prevalent and context-dependent, varying by flow regime, species, and assemblages. Our approach provides a robust framework for identifying thresholds at multiple scales, offering valuable insights to stakeholders for prioritizing conservation efforts and implementing targeted management actions for species of conservation concern.