Environmental DNA (eDNA) has emerged as a powerful tool for biodiversity assessment and monitoring which is enabling the detection of species from traces of genetic material present in environmental samples such as water, soil, or air. This method offers a cost-effective, non-invasive and highly sensitive alternative to traditional survey techniques which is critical in remote and logistically challenging environments such as those found across the Northern Territory (NT) of Australia. However, the application of eDNA for aquatic biodiversity monitoring in the NT is currently limited by the lack of a regionally relevant and taxonomically robust DNA reference libraries. Accurate species identification using eDNA data is entirely dependent on the availability and quality of publicly available reference sequences. To address this critical gap, this project will focus on generating a curated, high resolution genomic reference database for the freshwater and estuarine biodiversity with a focus on the Adelaide River system which is one of the NT’s ecologically significant catchments. The approach will involve linking DNA barcodes and mitogenome wide data to vouchered specimens that have been taxonomically verified by experts, this will ensure accuracy in species level identification. Ultimately, this work will enable reliable and standardised eDNA monitoring within the Adelaide River and serve as a foundational model for expansion to other NT catchments. It will support long term as well as scalable biodiversity monitoring frameworks critical to informing evidence-based conservation, environmental assessment and policy development across northern Australia.