Objective To detect the genotoxic effects of long-term administration of nano zinc oxide nanoparticles (ZnO NPs) particles on rats by using the alkaline comet assay in vivo. Methods In combination with the extended generation reproductive toxicity test, 13-week-old parental SD rats were administered orally with ZnO NPs at 0,7, 50, and 350 mg/kg (maximum concentration of the nanoscale dispersion), and body weight changes were recorded and observed. After 70 days, 10 male rats and 10 female rats were taken from each dose group and sacrificed. Ethylene diamine tetraacetic acid anticoagulated whole blood was taken from each rat. The comet assay kit was used for tableting, and the result of comet analysis were analyzed by quantitative analysis software (Casp). Results Compared with the vehicle control group, peripheral blood DNA damage cell rate and tail DNA of the high-dose group of male rats increased significantly, which were 28.60% and(36.38±5.84)%, respectively (P<0.05). Peripheral blood DNA damage cell rate and tail DNA of the high-dose group of female rats increased significantly, which were 27.31% and(18.80±2.96)%, respectively (P<0.05). Conclusion The result of alkaline comet assay in ZnO NPs were positive at 350 mg/kg group.