To investigate time- and spatial related variations in the composition of otter Lutra lutra diet, a total of 838 faecal samples was collected in the upper catchment of the Agri River (Basilicata region, southern Italy), and analysed. Data were split up according to the four seasons and between the main river and three of its tributaries. Fish and amphibians formed the bulk of otter diet, their consumption being inversely correlated. Trophic niche breadth was positively correlated with the frequency of occurrence of fish, whilst it was negatively correlated to that of amphibians and the altitude of the sampling stations. The frequency of consumption of fish did not vary through the year, whilst amphibians were mainly eaten during their hibernation and breeding period. Otter diet along the four main rivers differed significantly, the species being mainly piscivorous on the main river, whilst relying on alternative food resources on its tributaries, where habitat features or human interference reduced fish abundance. Fish availability seems to represent the main factor determining the composition and diversity of otter diet.