The genetic variability of three Gymnotus species from the Caracu stream, a small tributary of the left margin of Paraná River (Brazilian upper Paraná River floodplain), was estimated with data of 17 putative allozyme loci, which were obtained by using corn starch gel electrophoresis of 10 enzymatic systems: Aspartate aminotransferase (E. C. 2.6.1.1), Alcohol dehydrogenase (E. C. 1.1.1.1), Esterase (E. C. 3.1.1.1), Glucose dehydrogenase (E. C. 1.1.1.118), Glycerol-3-phosphate dehydrogenase (E. C. 1.1.1.8), Isocitrate dehydrogenase (E. C. 1.1.1.42), L-Lactate dehydrogenase (E. C. 1.1.1.27), Malate dehydrogenase (E. C. 1.1.1.37), Superoxide dismutase (E. C. 1.15.1.1) and Sorbitol dehydrogenase (E. C. 1.1.1.14). The genetic diversity was estimated as He = 0.3458 for G. pantanal, He = 0.2481 for G. inaequilabiatus, and He = 0.3152 for G. sylvius. The most divergent species were G. sylvius and G. pantanal (D = 0.117), and the most similar were G. inaequilabiatus and G. pantanal (D = 0.051). The data indicates that the observed genetic variability was very low and the expected variability estimated for these three species is very high, and the genetic differences among them are small. The data suggest that the process of speciation which produced these three species is recent.