Abstract: To establish a protocol of polyploid induction from multi-genotype seeds of wild L. concolor var. pulchellum in vitro, and develop a rapid, accurate and effective technique of ploidy confirmation, sterile in vitro germinating seeds obtained from 7 days' pre-cultivation was incubated in different concentrations of colchicine solution for different exposure times.Then their hypocotyl swollen was used as primary selection criteria of putative polyploids. The ploidy levels of putative polyploids were detected by flow cytometry combined with chromosome counts. The effects of different colchicine concentrations and exposure times on tetraploid production were compared. We have developed an seeds in vitro polyploid induction and identification protocol. The results indicated that the most efficient procedure for chromosome doubling was 0.10% cholchine treating the seeds for 36 hours, induction rate was 44.43%. The tetraploid plants were significantly different in color, thickness and surface of leaves and petiole as well as growth speed from diploid plants. Furthermore, the variations also happened within the different tetraploid genotype groups. Therefore, the technique of hypocotyl swollen of seeds combined with flow cytometry to detect polyploidy was efficient, this protocol provides a feasible method for inducing wild L. concolor var. pulchellum polyploids. Except for inducing L. concolor var. pulchellum polyploids, this in vitro polyploid induction process can also be used as a reference for doubling the chromosomes of other wild lily seeds.