Producing microbial oils via oleaginous yeast fermentation has drawn broad attention in the biodiesel industry. The oleaginous yeast Lipomyces starkeyi utilizing diverse carbon sources including glucose, xylose, glycerol, and willow wood sawdust (WWS) hydrolysate for the biosynthesis of oils in its cell growth were investigated in this study. High carbon/nitrogen ratios within the glucose media significantly increased the lipid content of Lipomyces starkeyi cells and modified the fatty acid composition of lipids, promoting the accumulation of C16:0 fatty acids and saturated fatty acids (C16:0 and C18:0). The accumulation of C18 fatty acids (C18:0, C18:1, and C18:2) and unsaturated fatty acids (C16:1, C18:1, and C18:2) was restricted. When crude glycerol and WWS hydrolysate were used as the sole carbon sources for L. starkeyi fermentation, the dry cell weight, lipid content, and lipid productivity were 9.1 g/L, 46.2%, and 4.2 g/L, respectively, for glycerol, and 8.2 g/L, 42.7%, and 3.5 g/L, respectively, for the hydrolysate solution. This study provides useful information for producing oils with L. starkeyi fermentation using glycerol and WWS hydrolysate as the primary or secondary carbon substrates.