In this work, we evaluated the short time-induced oxidative stress–mediated rapid metabolic and physiological responses of resistant and susceptible Sumatran fleabane [Conyza sumatrensis (Retz.) E. Walker; syn.: Erigeron sumatrensis Retz.] to 2,4-D herbicide. Under fixed conditions (25 C and 65 ± 5% relative humidity), we assayed injury symptoms, chlorophyll a fluorescence, and antioxidative systems of biotypes both resistant and susceptible to 2,4-D (1,005 g ae ha–¹). Under 15 versus 25 C temperatures and light and dark conditions, oxidative stress–mediated damage was assayed on plants that received 2,4-D herbicide applications. The injury symptoms observed in the 2,4-D–resistant biotype were rapid necrosis in leaves within 30 min, with the reestablishment of normal growth within 1 to 2 wk after 2,4-D treatment. The basal antioxidant enzyme activities of superoxide dismutase, catalase, and ascorbate peroxidase were greater in the resistant than in the susceptible biotype, although the activities of all enzymes generally did not differ between untreated and treated in the resistant biotype. The resistant biotype showed great reduction (at 1 and 4 h after application) in the photosynthetic electron transport chain performance index, while these metabolic changes were only detected after 4 h in the susceptible biotype. The resistant biotype recovered from the foliar damage 1 to 2 wk after 2,4-D application, while the susceptible biotype was controlled. The production of H₂O₂ was responsive to temperature and increased more rapidly in the 2,4-D–resistant biotype than in the susceptible one at both 15 and 25 C; however, there was a greater increase at 25 C in the resistant biotype. H₂O₂ production was not light dependent in 2,4-D–resistant C. sumatrensis, with increases even under dark conditions. The 2,4-D–resistant biotype showed rapid photosynthetic damage, possibly due to the rapid necrosis and leaf disruption, and increased H₂O₂ content compared with the susceptible biotype.