Reliable and comprehensive DNA reference data are a fundamental requirement for the success of eDNA metabarcoding analyses and molecular species identifications in Australian marine environments. The National Biodiversity DNA Library (NBDL) is a CSIRO-led multi-partner initiative to provide comprehensive DNA reference sequences for named Australian species, derived from expertly identified specimens held in Australia’s research collections. Starting with marine taxa, the NBDL is working in targeted campaigns to generate authoritative sequence data for taxonomic identification and make these data publicly available through a dedicated data and analysis portal. CSIRO has developed a cost-effective highly miniaturised genome-skimming technology ideally suited to collection specimens to complete this library. We are working in partnership with philanthropic and research organisations, Australian collections, state and federal governments, and with the guidance of taxon experts and the end-user community.