Nine hypostomine populations from Manso Reservoir (Rio Manso and Rio Cuiaba, upper Paraguay River basin, Brazil) were surveyed using allozyme electrophoresis. Three, sampled at the same sites, were not identified to the species level and were designated as Hypostomus sp. 1, Hypostomus sp. 2 and Hypostomus sp. 3. The survey resulted in 25 loci from 14 scored enzyme systems. Allozyme data allowed diagnostic marker recording for all species analyzed, including the undescribed taxa. Mean expected heterozygosity values were quite variable, ranging from 0.029 to 0.079. Hypotheses to explain this variation are discussed. The allelic frequencies and Nei's genetic identity values showed that Hypostomus sp. 1, Hypostomus sp. 2 and Hypostomus sp. 3 should be considered true biological species, and distinct from the other five Hypostomus species and Pterygoplichthys anisitsi. (C) 2007 Elsevier Ltd. All rights reserved.