The objective of this study was to evaluate toxicogenetic potential of surface water samples from rivers of center-west Brazil and analyze the influence of land use and cover and physicochemical parameters in genetic damage. Samples were collected during winter (June) and summer (November) at sampling sites from Dourados and Brilhante Rivers (Mato Grosso do Sul/Brazil). The toxicogenetic variables, including chromosomal alterations, micronuclei, and mitotic index, were analyzed in meristematic cells of Allium cepa; and micronuclei, nuclear abnormalities, and DNA strand breaks (arbitrary units, AUT) were analyzed in erythrocytes of Astyanax lacustris. The rivers presented physicochemical values outside the Brazilian laws, which can be a characteristic of human pollution (domestic sewage and local agriculture). The results of A. cepa test suggest that the water samples from Dourados and Brilhante rivers exerted significant (p < 0.05) cytotoxic and genotoxic effects, in both periods of collection, especially alterations in mitotic index. In blood cells of A. lacustris, genotoxic effect of the water samples from the rivers also was observed as significant nuclear abnormalities, DNA breaks (UAT), in both sampling periods, compared with the negative control. Spearman correlation analyses revealed that data of land use and cover and physicochemical parameters were statistically correlated with DNA damages in bioassays. This study demonstrates toxicogenetic potential of water samples from Dourados and Brilhante rivers; furthermore, the type of land use and land cover and physicochemical parameters were revealed to have influence on toxicogenetic damage.