Hypostomus hermanni (Ihering, 1905) and three other morphotypes of the genus Hypostomus were collected from the Ribeirão Keller, a small tributary of Rio Ivaí, a tributary of upper Rio Paraná. An allozyme analysis of 25 gene loci revealed diagnostic loci alleles for each population and there were fixed differences between some of them at some loci. Thus all populations were genetically distinct, although there were many common alleles. Heterozygosity (He) ranged from 0.059 in Hypostomus sp. 2 to 0.144 in Hypostomus sp. 1 and was higher than the average for other species of Hypostomus and also for fish in general. H. hermanni and Hypostomus sp. 1 were more similar to each other (I = 0.878) whereas H. hermanni and Hypostomus sp. 2 showed the least genetic identity (I = 0.392).