Coenzyme Q₁₀(CoQ₁₀ ) is an important electron transfer molecule in the respiratory chain and the indispensable coenzyme in the production of ATP, and it is widely used in therapeutic applications for several diseases such as heart diseases, hepatitis, Parkinson and so on. In order to improve the production of CoQ₁₀, this paper studies the screening of the mutant Rhodobacter Sphaeroides aiming for a higher CoQ₁₀ production by genome shuffling. According to the biosynthetic pathway and its mechanism, various resistance markers are selected, including the roxithromycin, the kanamycin, the p-hydroxy benzcic acid, the vitamin K3 and sodium sulfide(Na₂S). Their concentrations are determined according to the tolerance experiments on Rhodobacter sphaeroides. The improved starting population including nine mutant strains is generated by different resistance markers and different mutagenesis ways, such as the ultraviolet irradiation, the ultraviolet/lithium chloride, the diethyl sulfate, the microwave radiation and the γ Co60. Several high CoQ₁₀-producing colonies, including PN13, are selected from the first shuffled library. The CoQ₁₀ content of the PN13 reaches 2.39mg/g, 2.52 times of that of the wild-type strain.