AbstractThe rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo truttaL.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site‐associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non‐target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.