Using goldfish (Carassius auratus) as the model animal, the present study revealed the types of the DNA damage induced by monocrotophos, a highly toxic organophosphorus pesticide, and explored the mechanism underlying the DNA-damaging effect of this pesticide. Results of the alkaline comet assay showed that global DNA damage (including single- and double-strand breaks and alkali-labile sites) in peripheral erythrocytes of goldfish, measured as olive tail moment, was significantly increased by exposure to 0.01, 0.10, and 1.00 mg/L monocrotophos for 24, 48, 96, and 168 h. In particular, alkali-labile sites rather than single- or double-strand breaks, distinguished by the alkaline, pH 12.1, and neutral comet assays, were mainly induced by monocrotophos at 48 h. Oxidative damage in DNA bases and telomeric DNA was investigated by using the alkaline comet assay combined with endonuclease III or formamidopyrimidine DNA glycosylase and with fluorescence in situ hybridization, respectively. Further, glutathione peroxidase activity significantly decreased at 24 h but increased at 96 and 168 h, and malondialdehyde concentrations significantly increased at 48 h but gradually decreased at 96 and 168 h, which indicated an over-production of reactive oxygen species (ROS) at short exposure durations, but effective scavenging at long exposure durations in the peripheral blood tissues. Accordingly, our results suggest that DNA damage induced by monocrotophos in fish blood cells is possibly due to the inhibition of ROS scavenging and resulted accumulation of ROS.