<p dir="ltr">Herbicide selectivity in forest plantations remains insufficiently understood, particularly regarding the physiological and genetic mechanisms underlying tolerance to HPPD inhibitors. <i>Eucalyptus urograndis</i>, one of the most widely planted forest species in Brazil, is routinely exposed to herbicide drift, yet its biochemical and genetic responses to isoxaflutole have not been fully characterized. This study examined whether tolerance to isoxaflutole is mediated by differential metabolic detoxification and genetically structured variation among open-pollinated progenies derived from three commercial clones (I-144, IPB1 and Suza217). Leaf metabolite dynamics were quantified via HPLC following applications of 200 and 400 g ha⁻¹ isoxaflutole, measuring levels of the parent compound (Isoxa), the active metabolite diketonitrile (DKN), and the detoxification product benzoic acid (BA). Dose–response assays (0–32× the commercial rate) evaluated phytotoxicity and biomass, while genetic parameters for injury were estimated using mixed models. Genetic diversity and inbreeding were assessed using 21 SSR markers. Isoxa dissipated rapidly in all progenies, with early peaks followed by progressive increases in DKN and late accumulation of BA. Suza217 and I-144 demonstrated greater metabolic conversion of DKN to BA than IPB1, indicating more efficient detoxification. No consistent dose-dependent reduction in biomass was observed, even at 32× the recommended rate, evidencing inherently high tolerance. Damage severity exhibited moderate to high broad-sense heritability at later assessments, revealing a strong genetic component. SSR analyses showed moderate to high genetic diversity, with IPB1 displaying the greatest allelic richness and Suza217 the highest excess heterozygosity. These results demonstrate that isoxaflutole tolerance in <i>E. urograndis</i> is primarily driven by metabolic detoxification capacity and supported by substantial genetic variability, providing a foundation for breeding herbicide-tolerant planting stock.</p>
