Abstract: Microscopic hair identification is a non-invasive, simple, and economical method applied in scientific studies to identify mammal species. In ecology, this method is used mainly in mastofaunistic inventories and dietary studies. In the last decade, the number of dietary studies using the microscopic identification of hairs has grown substantially, but the application of this technique as a tool for the identification of both predators and prey species is still scant. Thus, the aim of

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... his study was to identify predator and prey hairs in scat samples from the two largest species of carnivores in the Neotropical region, the jaguar (Panthera onca Linnaeus, 1758) and the puma (Puma concolor Linnaeus, 1771). We examined a total of 100 scat samples being 50 from the Pantanal of Mato Grosso do Sul and 50 from the Atlantic Forest of Paraná. We used different identification categories that included the hair microscopic and macroscopic identification, as well as the use of hooves and nails present in the scats associated with tracks and kills found in the field. We identified 57 prey items in the Pantanal samples and 61 in the Atlantic Forest samples. Predator´s hairs were identified in 34% of Pantanal samples and in 46% of Atlantic Forest samples. The combination of hair microscopic and macroscopic characteristics was efficient in the identification of different taxonomic levels, with most identifications reaching the level of the species. However, the methodological protocol for microscopic hair identification was not fully effective in obtaining all the microstructural patterns of the studied mammals. Adjustments in the technique are necessary to differentiate microstructural characteristics of species belonging to the same family. We recommend macroscopic identification of scat content items (hairs, hooves or nails) of both prey and predators to be used to complete the microscopic hair identification technique in dietary ecological studies.