Aquatic invasive plant species pose an urgent and growing threat to freshwater ecosystems in the United States, displacing native sportfish biodiversity, degrading ecosystem function, and imposing escalating economic and ecological costs. Globally, aquatic invasive plants have caused over $32 billion in the last five decades, with 65% of these costs attributed to freshwater ecosystems. In the United States alone, annual expenditures for aquatic plant control approach $100 million, underscoring the immense financial burden of these invasions. Despite the ongoing financial investment, current molecular tools lack the resolution to reliably distinguish closely related invasive plant species, limiting effective early detection and rapid response. Building on our successful foundational project, “Improving Conservation Efforts for Sportfish and Waterfowl: Developing eDNA Tools for Use in Aquatic Invasive Species Detection,” which applied chloroplast-based nanopore sequencing but revealed critical taxonomic ambiguities, this proposal advances to a transformative next phase. We will generate high-quality nuclear genome assemblies for 5 high-priority aquatic invasive plant species – including Nitellopsis obtusa (starry stonewort), Myriophyllum spicatum (Eurasian watermilfoil), and Hydrilla verticillate using PacBio HiFi long-read sequencing and the Minnesota Supercomputing Institute’s cutting-edge bioinformatics resources. Our primary objective is to identify highly specific, species-level nuclear genetic markers that provide unprecedented discriminatory power. These markers will underpin scalable, field-ready molecular assays such as high-throughput quantitative PCR (HTqPCR) and loop-mediated isothermal amplification (LAMP), essential for advancing eDNA surveillance and empowering the early detection and rapid response framework for aquatic invasive plant species in the United States. The whole genome sequence and species-level nuclear genetic markers will be shared via open access websites (National Center for Biotechnology Information, NCBI) enabling other researchers to freely build upon this work. Delivering these foundational genomic resources is critical to protecting freshwater biodiversity, supporting healthy populations of sportfish, reducing costly invasions, and ensuring long-term ecological and economic resilience.