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17α-Methyltestosterone (MT) is widely used in fish hatcheries of many countries to produce male monosex populations. Its genotoxic risk to fish species is not well known and studies in other in vivo models are still inconclusive. MT was tested for genotoxicity in the fish species Oreochromis niloticus (tilapia), a target species, and Astyanax bimaculatus (lambari), a native non-target species. Genotoxicity was evaluated by the micronucleus test (MN), nuclear abnormalities (NA), and comet assay using peripheral erythrocytes of both species after a 96-h exposure to MT at concentrations of 0.01, 0.1, and 1.0 mg/L in the water. At the lowest exposure level of 0.01 mg/L, MT induced MN in both species and NA only in O. niloticus. These effects were not observed in the comet assay. Chromatographic analysis of water samples collected from aquariums at the beginning and end of each experiment showed that MT was consumed during the 96-h exposure. At the highest level of exposure (1.0 mg/L), 81.69% of the hormone was consumed during the exposure period. The chromatogram showed that at the lowest concentration level of 0.01 mg/L, 99.56% MT was consumed by the end of the exposure period. Thus, exposure to MT did not cause genotoxicity in either fish species.