The analysis of cuticle and medulla hair microstructure is a simple and inexpensive technique to identify mammal species for a variety of applications. We studied the guard-hairs of 66 individuals of eight felid species occurring in Brazil (Leopardus colocolo, L.geoffroyi, L.pardalis, L.tigrinus, L.wiedii, Panthera onca, Puma concolor, Puma yagouaroundi), through hair samples collected from anesthetized zoo animals. The microstructure of the guard-hairs was analyzed and described through cuticle impressions and medulla preparations; a blind test was conducted to evaluate the accuracy of species identification. Although distinctive morphological characters could be identified for each species, the subtlety of these characters and the overlap of features among species resulted in a relatively poor accuracy (75%). The identification of pairs or trios of species whose hair has similar morphologies (Group A: L. pardalis, L. tigrinus, L. wiedii; Group B: L. colocolo, L. geoffroyi, P. yagouaroundi; Group C: P. concolor, P. onca) significantly improved accuracy (91%). The identification of Brazilian felids through the microstructure of their hair is challenging and requires careful examination of subtle features, and should be complemented by more accurate techniques and/or be limited mostly to applications where high accuracy is not essential or where a broader taxonomic scale is being evaluated.