Abstract: In order to obtain quinclorac-resistant tobacco breeding materials, different concentrations of quinclorac were sprayed on tobacco to analyze the change pattern of leaf area and the damage of tobacco, confirm the critical concentration as well as optimal observation time for tobacco seedlings after quinclorac treatment. Meanwhile, the saturated spray method was conducted on tobacco EMS mutant library to identify resistance lines on a large scale. The results show that the thresholds of spraying with quinclorac are 0.5 mg/L. The herbicide damage symptoms were observed obviously in tobacco leaves once the application concentration is beyond the critical value. To obtain highly resistant materials, the optimum concentration of quinclorac was 1 mg/L, and observation time were set as 14 days after application. Moreover, we obtained many mutant resources with high, medium resistance and sensitivity to quinclorac treatment. In addition, the high resistance of QR-19 lines to quinclorac in F1 generation might be controlled by a recessive gene. The research provides a material basis for herbicide resistance breeding and demonstrating the mechanism of auxin-herbicide resistance.