The genotoxic activity of surface water samples from four sites along the Paraguay River, near Cáceres, Mato Grosso State, Brazil, was investigated using the Drosophila melanogaster Somatic Mutation and Recombination test (SMART). Effluents from sanitary sewers and agroindustrial effluents (residual effluents from slaughterhouses, leather tanneries, and dairies) flow into the Paraguay River, and directly or indirectly contaminate water from sampling sites 1-3. Site 4 was an upriver reference site that received no domestic or agroindustrial discharges. Water was collected at 4 time periods: September 2003 and August 2004 (periods of low water or drought); and April 2004 and March 2005 (periods of high water or flood). Chromium concentrations above statutory limits were detected at sites 1-3 (August 2004), and sites 1, 2 and 4 (March 2005). Sulfur compounds were also detected at sites 1-3. The SMART performed using standard (ST) cross flies detected genotoxic responses in only two samples, the August 2004 site 1 sample and the March 2005 site 2 sample. Many more samples were positive using high bioactivation (HB) cross flies: site 1 (all collection periods), site 2 (September 2003 and April 2004), and site 3 (September 2003 and August 2004). Mutant frequency comparisons between marker-heterozygous and balancer-heterozygous flies from the HB cross indicated that the positive genotoxic responses for the site 2 (April 2004) and site 3 (September 2003) samples were due mainly to mitotic recombination. Our findings indicate that the section of the Paraguay River within the urban perimeter of Cáceres is contaminated with genotoxic agents.