The United States east coast population of the gray seal (Halichoerus grypus (Fabricius, 1791)) was once hunted to near extirpation, but the population has since rebounded due to protection by the Marine Mammal Protection Act of 1972. Although this population growth is seen as a success by conservationists, others are concerned about the economic and ecological impact of gray seals on New England fisheries. The study objective was to quantify flatfish presence in the diet of gray seals using an analysis of prey DNA in seal scat (molecular scatology). This may reduce a potential bias in other diet analysis methods since flatfish may not be swallowed whole, and therefore, their identifying otoliths may not be present. Scats were collected from Muskeget and Monomoy islands off Cape Cod, Massachusetts, USA, in 2016, 2017, and 2018 and analyzed for flatfish presence using species-specific polymerase chain reaction (PCR) assays and a hard parts analysis using otoliths. Frequency of occurrence for flatfish species from DNA was higher than estimated in previous literature on gray seal diet and a concurrent otolith analysis of the same samples, suggesting that previous analyses may have potentially underestimated the importance of flatfish in the diet.