Various contaminants are present in aquatic environment and pose potential threats to pelagic and benthic organisms, calling for effective risk assessment. Traditional risk assessments based on target analysis are useful when the principal contaminants responsible for ecological risk are known; however, these approaches become challenging when dealing with chemical mixtures. In addition, the compositions of chemical mixtures often differ in regions with different levels of socioeconomic development, requiring risk assessment methods that are applicable under different pollution scenarios. Herein, in situ bioassays were conducted with two native species, Chinese rare minnows (Gobiocypris rarus) and Asian clams (Corbicula f luminea), in economically developed watersheds in China (Pearl River Basin (PRB) and Taihu Lake Basin (THB)) and agriculture-dominated Poyang Lake Basin (PYB). Significant lethal and sublethal effects (e.g., neurotoxicity, reproductive toxicity, and metabolic and oxidative stress) were observed in fish and clams irrespective of economic gradients. Notably, ecological effects differed significantly between water and sediment phases within the same region. Target (98 contaminants) and suspect screening (942 contaminants) revealed regional-specific characteristics. Ecological risk assessments using a weight of evidence approach demonstrated that both water and sediment in the PRB were at moderate to high risk, as was the sediment in the less developed PYB. However, the characteristics of mixture pollution varied greatly among regions. Suspect screening identified many pollutants that are not regularly monitored but are present at high environmental concentrations and are linked to local industrial production. These distinct mixture risk characteristics across different basins suggest that mitigating aquatic pollution requires region-specific management measures.