Communities of zooplankton can be adversely affected by contamination resulting from human activities. Yet understanding the influence of water quality on zooplankton under field-conditions is hindered by traditional labor-intensive approaches that are prone to incomplete or uncertain taxonomic determinations. Here, for the first time, an eco-genomic approach, based on genetic diversity in the mitochondrial cytochrome c oxidase I (COI) region of DNA of zooplankton was used to develop a site-specific, water quality criterion (WQC) for ammonia (NH3). Ammonia has been recognized as a primary stressor in the catchment of the large, eutrophic Tai Lake, China. Nutrients, especially NH3 and nitrite (NO3-) had more significant effects on structure of the zooplankton community than did other environmental factors. Abundances of rotifers increased along a gradient of increasing concentrations of total ammonia nitrogen (TAN), while abundances of copepods and cladocera decreased. A novel, rapid, species sensitivity distribution (SSD) approach based on operational taxonomic units (OTUs) was established to develop a WQC for NH3. The WQC based on OTUs was consistent with the WQC based on the traditional morphology taxonomy approach. This genetics-based SSD approach could be a useful tool for monitoring for status and trends in species composition and deriving ecological criteria and an efficient biomonitoring tool to protect local aquatic ecosystems in virtually any aquatic ecosystem.