AbstractPeruvian watergrass (Luziola peruviana J.F. Gmel.) is a perennial weed often found in paddy fields in the southern region of Brazil. Its chemical control is performed mainly by herbicides such as glyphosate in burndown, whose efficacy depends on soil moisture. Under excess‐water soil conditions, glyphosate efficacy is reduced, probably due to reduced translocation and the involvement of photoassimilates partition. Furthermore, little is known about the stress tolerance of these plants subjected to water deficit. Based on that, we performed experiments to verify the effect of soil moisture on the biochemical parameters of Peruvian watergrass and glyphosate translocation to the stolon and roots. Total soluble sugars, sucrose, starch, and glyphosate concentrations in leaves, culms, roots, and glyphosate partitioning were quantified. The malondialdehyde content and the lipid peroxidation increased with the drainage. Under drainage conditions, superoxide production was higher in roots than leaves, while peroxide production was lower in roots than leaves. The electrolyte leakage increased with the drainage and glyphosate application. The total soluble sugar content varied in each organ with a significant increase in the culm 14 days after application in the draining treatment. Sucrose varied considerably and reached its maximum concentration at 21 days after herbicide application. The starch accumulation in the culm was 7 days after applying the highest glyphosate dose. The presence of glyphosate in the roots subjected to soil drainage indicates the movement of this herbicide. In conclusion, soil drainage profoundly impacts Peruvian watergrass physiology, increasing the photoassimilates and glyphosate translocation to the stolon and root. This explains why glyphosate efficacy is higher under drainage conditions.