In this study, we use whole-genome sequence data to identify diagnostic SNPs for the development of CRISPR-based SHERLOCK assays capable of rapidly, accurately, and sensitively distinguishing ESA-listed Chinook salmon run types (winter- and spring-run) from each other and from unlisted run types (fall- and late fall-run), in California Central Valley. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent to transform how conservation scientists and managers view genetic identification going forward.