Molecular techniques offer sensitive, specific, noninvasive monitoring of target species from a variety of environmental samples. We recently developed a CRISPR‐Cas‐based eDNA assay for rapid single‐species detection as a route to a simple, cost‐effective biosensor device. CRISPR‐Cas‐based diagnostic assays use isothermal conditions in combination with a highly specific sequence recognition system. This CRISPR‐Cas assay was designed to target Salmo salar, and we previously demonstrated its utility in eDNA samples from sites in Ireland. The aim of this study was to validate our assay in two larger sample sets from Canada (n = 16/n = 63) in comparison with an independent S. salar qPCR assay. We demonstrate that overall, the CRISPR‐Cas assay performs similarly to qPCR for assessing the presence or absence of S. salar from eDNA and provides a viable alternative approach where qPCR assay design and application have proven to be challenging.