Abstract: Taking two kinds of male sterile lilies ‘5-4’ and ‘5-35’ with good color, flower type and excellent growth as test materials, tissue culture methods were used to study different disinfection times, hormone concentrations and different substrate formulation on the tissue culture process of two kinds of male sterile lilies, in order to establish their rapid propagation system and provide reference for mass production of non-pollen lilies.The results showed that the best way to sterilize explants was 75% ethanol disinfection for 20 seconds, 10% NaClO solution for 8 minutes, and 75% ethanol for 30 seconds, 10% NaClO solution for 6 minutes; the best induction medium was MS+NAA 1.0 mg·L^-1+6-BA 0.5 mg·L^-1 and MS+NAA 1.5 mg·L^-1+6-BA 0.5 mg·L^-1;the best proliferation medium was MS+6-BA 1.5 mg·L^-1+NAA 0.5 mg·L^-1;the best rooting medium was 1/2 MS+IBA 0.1 mg·L^-1+NAA 0.1 mg·L^-1+sucrose 40 g·L^-1 and 1/2 MS+IBA 0.2 mg·L^-1+NAA 0.1 mg·L^-1+sucrose 40 g·L^-1;the best transplanting substrate ratio was vermiculite∶perlite∶peat=1∶1∶1.