The Eurasian otter Lutra lutra suffered a sharp decline in the last century caused by multiple pressures, including human persecution, habitat loss, and water pollution. In Italy, the species is currently listed as Vulnerable in the Italian Red List. For top predators a sound knowledge of the feeding ecology is essential to adopt effective conservation strategies, especially at local scale. We analysed the trophic niche of the Eurasian otter in three rivers located in the core area of the Italian otter range and compared niche width and prey composition among river sectors. Prey remains were examined in 415 spraints collected from July to October 2019 at 17 frequent marking sites along seven river sectors (one upstream, three medium course, and three downstream). Differences in the probability of occurrence of five prey categories (fish, crustaceans, amphibians, reptiles, and insects) were compared among rivers and river sectors through Generalized Linear Mixed Models. Pianka index (Ojk) was used to evaluate the trophic niche overlap among rivers sector pairs. Results showed that fish was the main prey in the whole study area, followed by crustaceans, amphibians, insects, and reptiles. However, prey remains in spraints reflected prey composition at very local scale, with fish prevailing in all downstream sectors, while amphibians, crustaceans, and insects mainly represented in the middle sectors. The endemic crayfish Austropotamobius italicus was almost the unique prey in the single upstream investigated. Accordingly, trophic niche differed more among river sectors within the same river (Ojk = 0.21-0.28) than among same sectors (i.e., downstream and middle course) of different rivers (Ojk > 0.6). This spatial pattern is discussed both in terms of behavioural ecology and digestive physiology of the Eurasian otter.