This investigation assessed the interaction of surface water samples with DNA to quantitatively and qualitatively characterize their mutagenic and/or recombinagenic activity. Samples were obtained at three different sites along the Tocantins River (Tocantins State, Brazil). The area has withstood the impact mainly of rural activities, which release different chemical compounds in the environment. The Drosophila melanogaster Somatic Mutation and Recombination Test (SMART) was performed in standard (ST) and high bioactivation (HB) crosses. SMART is useful for the detection of mutational and recombinational events induced by genotoxins of direct and indirect action. Results demonstrated that samples collected in both seasons were able to induce increments on the mutant spot frequencies in the larvae of the HB cross. Genotoxicity was related to a massive recombinagenic activity. The positive responses ascribed to only the HB cross means that it is linked to pro-genotoxins requiring metabolic activation. The SMART wing test in Drosophila melanogaster was shown to be highly sensitive to detect genotoxic agents present in the aquatic environment impacted by agriculture.