Three Brazilian populations of the armored catfish Hypostomus regani (Ihering, 1905) were sampled, one from the Corumbá Reservoir in Goiás state, another from the Itaipu Reservoir in Paraná state and a third from the Manso Reservoir in Mato Grosso state. Allozyme electrophoresis was used to establish the genetic structure of the species, with the analysis of liver, heart and muscles tissues allowing the scoring of 25 loci from 14 enzymatic systems. Although no diagnostic loci were found, some exclusive rare alleles were recorded for the three populations. The genetically most similar populations were those from Corumbá and Itaipu, and the most distant were the populations from Manso and Corumbá. The allozyme data showed three structured populations belonging to the same species H. regani (F ST = 0.173).