Based on molecular markers (COII and ITS1-ITS2) and morphological data, we describe three new Neotropical species of Gyrodactylus von Nordmann, 1832 from Scleromystax barbatus (Quoy et Gaimard) and Scleromystax macropterus (Regan) from southern Brazil. The three new species can be distinguished from each other by sequences of both molecular markers and morphology of hooks and anchors. Gyrodactylus bueni sp. n. is characterised by having hook with shaft curved, heel straight, shelf straight, toe pointed, anchor with superficial root slender, elongate and male copulatory organ armed with two rows of spinelets. Gyrodactylus major sp. n. presents hook with shaft, point curved, proximal shaft straight, heel convex, shelf convex, toe concave, anchor with superficial root robust and male copulatory organ armed with two rows of spinelets. Gyrodactylus scleromystaci sp. n. presents hook with shaft, point recurved, heel convex, shelf convex, toe pointed, anchor with superficial root curved and male copulatory organ armed with two rows of spinelets. These species appear to be closely related to other species of Gyrodactylus known from other species of Callichthyidae. These new species, however, differ by the comparative morphology of the haptoral hard structures and molecular data. Comparative analysis of sequences from these species of Gyrodactylus suggests that the COII gene may represent an important marker for the taxonomy of species of Gyrodactylidae and, perhaps, for species of other lineages of Monogenoidea.