The blood infection by Trypanosoma sp. in tuvira (Gymnotus aff. inaequilabiatus) from the Pantanal wetland was reported in this study. Ten fish from the Paraguay River in the Pantanal were evaluated for the presence of hemoflagellates. Trypomastigotes of Trypanosoma sp. were observed in blood smears from three fish (30% prevalence) and some forms were seen to be undergoing division. Using the diagnostic methods of fresh examination and blood centrifugation in hematocrit capillary tubes, the prevalence rate was 80%. This is the first report of Trypanosoma sp. in tuvira in Brazil.