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Glyphosate is an agrochemical widely used to control weeds. However, glyphosate spreads to water bodies by spray-drift, run-off and leaching, potentially causing detrimental effects on non-target biota. There is no information on the occurrence of this herbicide in water bodies near crop fields in Mexico, although it is the most commonly used pesticide in this country. To fill this gap, we quantified glyphosate in water bodies from twenty-three locations, including natural protected areas and agricultural areas in southern Mexico, during the dry and the rainy seasons. We expected (1) higher concentrations during the dry season due to reduced dilution by precipitation and, (2) absence of glyphosate in the protected areas. In agreement with our expectation, concentration of glyphosate was higher during the dry season (up to 36.7 μg/L). Nonetheless, glyphosate was detected in all samples-including natural protected areas. These results emphasize the need for an evaluation of the impact of glyphosate on native species as well as regulate its use. Inglés