Microbial lipids are valuable renewable precursors for fuels, chemicals, and nutraceuticals. However, a major limitation to lipid production from cellulosic biomass is low lipid titers, largely stemming from difficulty in preparing highly concentrated biomass hydrolysates and further converting such hydrolysates by microbes. In this study a new process was proposed for high-titer lipid production. In Particular, a concentrated biomass hydrolysate with 121.7 g/L monomeric sugars (113.5 g/L glucose and 8.2 g/L xylose) and 88% glucose yield was prepared from switchgrass pretreated by an acidic aqueous choline chloride: ethylene glycol (ChCl:EG) deep eutectic solvent (DES) within only 24 h of enzymatic hydrolysis. The oleaginous yeast Lipomyces tetrasporus (NRRL Y-11562) was able to consume both glucose and xylose in the biomass hydrolysate and produce 17.6 g/L lipid in batch fermentation. This study demonstrated a new process enabled by DES pretreatment for the production of microbial lipids at high titer from lignocellulosic biomass.