Assessment of pinniped predation most often relies on analysis of the hard, undigested prey remains evident in faecal (scat) samples. For many prey species this method can yield valuable information on predator–prey interactions. For some genera, however, species diagnostic characteristics are lost during the process of prey digestion, thereby preventing morphological identification of fish prey species. Here, the feasibility of using faecal DNA to detect the presence of salmonids in pinniped scat samples and to distinguish reliably between sea trout Salmo trutta and Atlantic salmon S. salar was assessed. Novel salmonid mitochondrial DNA (mtDNA) primers were designed to amplify 162 bp of the 16S rDNA and a 327 bp section of the cytochrome b gene. Species-specific banding patterns were obtained by digestion of the cytochrome b PCR product with the restriction endonuclease AluI, and confirmed by the species-specific amplification of the 16S rDNA fragment from Atlantic salmon. Scats collected from captive grey seals Halichoerus grypus fed on known monospecific diets used to validate the PCR-RFLP assay indicated a probability of at least 95.8% (23 of 24 faecal extracts) of detecting salmonids using DNA extracted from the scat matrix. Implemented alongside conventional prey remains analyses, this technique presents a promising new method for examining prey composition and assessing pinniped predation on salmonids.