Abstract Aim To present a fish fauna survey from rivers draining the south-western region of the Serra da Mantiqueira, and to investigate the effectiveness of that mountain range as a biogeographical barrier isolating the fish faunas of the Paraíba do Sul basin and the upper Paraná river. Location Six drainages from the south-western portion of the Serra da Mantiqueira in the region of Campos do Jordão, Brazil, were studied. They included three drainages belonging to the Paraíba do Sul basin (Piracuama, Grande and Buenos), and three belonging to the Sapucaí basin, Rio Paraná system (Sapucaí-Mirim, upper Sapucaí and Santo Antônio). Methods The survey was conducted in two sampling trips supplemented by examination of specimens preserved in museum collections. Parsimony analysis of endemicity (PAE) was used to detect the hierarchy of relationships among the six drainages. Results A total of 47 species of fishes was recorded in the six drainages. The main watershed divide is an effective barrier between the two basins, with 28 species occurring exclusively in the Paraíba do Sul versant, and 15 occurring exclusively on the slope of the Sapucaí basin. The PAE of 18 species with cladistically informative distributions and unproblematic taxonomic diagnoses produced a single area cladogram, with complete congruence among 14 species. Main conclusions The main Mantiqueira watershed divide is an effective biogeographical barrier isolating the fish faunas of the Paraíba do Sul and the Sapucaí basin (upper Paraná system). Previous hypotheses of a past connection between the upper Paraná and the Paraíba do Sul are not supported by extant species distributions in the Serra da Mantiqueira region. Within-basin drainage differentiation, however, is relatively low.