ABSTRACT Applied eDNA metabarcoding is increasingly being used to generate actionable results to inform management decisions, regulations, or policy development. Because of these important downstream considerations, optimizing workflow elements is now essential to increasing standardization, efficiency, and confidence of metabarcoding results. Reference DNA sequences are critical workflow elements that currently lack consistent approaches to generating, curating, or publishing. Here we present a complete (mitochondrial genome and nuclear ribosomal DNA cistron) and high quality reference DNA sequence library for the freshwater fishes of British Columbia, Canada. This resource is published as the Novel Applied eDNA Metabarcoding Reference Sequences (NAMERS) repository ( https://namers.ca ), a user-friendly and interactive website for specialists and non-specialists alike to explore and generate custom reference libraries for taxa and genes of interest. We demonstrate the power of NAMERS to optimize applied eDNA metabarcoding workflows at the study design stage by analyzing the number of primer mismatches and resolution power of existing metabarcoding markers. To meet the increasing demand for actionable eDNA metabarcoding applications, NAMERS demonstrates that high quality curated genomic information is within a reasonable reach. It is timely to establish this framework as the new gold standard and coordinate our efforts to generate this type of reference data at scale.