The Chinese government implemented the ambitious south-north water transfer project (SNWTP), which aims to transport water from the Yangtze to the north of China where water shortages are severe. Although the ecological impacts of this project have been addressed publically, there remains a poor understanding of the effects of such large-scale water transfers on the populations of aquatic species. The potential ecological impacts of such water transfers on the Hongze Lake fish assemblages are assessed here using Self-Organizing Map (SOM) and Random Forest (RF) modeling. Using SOM, twenty-three fish species in 15 sampling sites were classified into two assemblages and four sub-assemblages corresponding to four distinct habitats (deep water macrophytes, deep water bare silt, shallow water bare silt, and shallow water macrophytes). The RF model further showed that water depth and transparency were the abiotic drivers underpinning fish assemblages in Hongze Lake. As the SNWTP is forecasted to modify the distribution of water depth and water clarity, major knock-on effects are expected on downstream lake fish assemblages.