Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms—epimastigotes and short trypomastigotes—were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.