Abstract: To study the mutagenic effects of fast neutron irradiation (FNI) on tobacco traits and to investigate the feasibility of constructing a flue-cured tobacco mutant library with mutagenesis method, the effects of FNI at different doses (within the range of 5-20 GY) on the germination rate, seedling growth and seedling rate of K326 were studied. The results showed that:1) FNI was highly efficient in causing mutation in tobacco, and various mutants were obtained. It was a feasible method to construct a mutant library. 2) The appropriate dose of FNI for K326 seeds ranged from 10 to 15 GY. 3) The agronomic traits of fast neutron-mutagenized populations were various, and the populations could be used to study functional genomics or tobacco breeding as germplasm resources. Thus, FNI is an effective, reliable and rapid method for constructing a tobacco mutant library.