Abstract: Objective:Exploring the optimum conditions of the genome shuffling which applied to the key steps of Rhodotorula. Method:With Rhodotorula cp-2-11、cp-2-6、cp-28 as the tested material, we explored the effects of several factors including cell age, enzyme concentration, hydrolysis time, inactivation conditions, fusion time on genome shuffling. Results:The protoplast preparation rate and regeneration rate was supreme when the cell age was 12 hours, the enzyme concentration was 2% and the hydrolysis time was 70 minute.15W UV light, the irradiation distance was 35cm irradiates 18 minute and 60℃ water bath inactive 35 minutes were used as the conditions of Inactivation, fused 23 minute in 35% PEG as the fusion condition. Conclusions: Through determining the optimum conditions of genome shuffling, the follow-up work was improved effectively. After several rounds of genome shuffling, the high yield strains were breed, which increased by 122% than original strains.