Glyphosate is a popular herbicide used worldwide, and several studies consider it to be an environmental hazard affecting human health. The present study aimed to detect glyphosate in six different reservoirs of Paraíba do Sul and Guandu River Basins in Southeast Brazil, used for multiple purposes, including fishery activities and domestic water supply. Ion chromatography was used to analyze the water samples, as it is a fast and environmentally friendly technique to detect glyphosate. Our results revealed that, despite differences related to trophic state, season of the year or distance to urban areas, glyphosate was detected in all reservoirs and in three of them with concentrations above the limit imposed by Brazilian legislation. Among the environmental variables studied, turbidity presented the highest correlation with glyphosate concentrations. The effect of rainfall increasing turbidity in the rivers reinforces the importance of draining waters from surrounding areas that transport glyphosate into the aquatic ecosystems. The detection of the herbicide in the various systems confirms the wide use of this compound in the drainage basins of the studied reservoirs and highlights the importance of water monitoring. Further, the results reveal how urgent and important it is to explore through laboratory experiments the pathways of degradation of this herbicide in tropical and subtropical aquatic environments together with its effects on flora and fauna.