Crude glycerol, discharged from biodiesel production process, is a potential carbon source for microbial lipid production. The capability of using crude glycerol as sole carbon source for microbial lipid production by oleaginous yeasts Trichosporon fermentans and Trichosporon cutaneum was investigated for the first time. T. fermentans and T. cutaneum could use crude glycerol for efficient lipid production, and the optimal glycerol concentration for which were 50 and 70 g/L, respectively. The optimum nitrogen source, C/N, inoculum concentration, and pH were yeast extract + peptone, 60, 10.0%, and 6.0, respectively. The most suitable culture temperature for T. fermentans and T. cutaneum were 28 and 30 degrees C, respectively. Under the optimal conditions, the maximum biomass, lipid content, lipid yield, and lipid coefficient of T. fermentans and T. cutaneum were 16.0 g/L, 32.4%, 5.2 g/L, and 16.5%, and 17.4 g/L, 32.2%, 5.6 g/L, and 17.0%, respectively. Moreover, it was found that methanol present in the crude glycerol had minor influence on the lipid production. Addition of surfactant potassium oleate into the medium could slightly stimulate the cell growth and lipid accumulation of both yeasts. This study shows that T. fermentans and T. cutaneum are promising strains for lipid production on crude glycerol.