Trichosporon oleaginosus is one of the most promising hosts for microbial lipid production owing to its high-productivity; In an effort to develop an economical production process, we engineered T. oleaginosus towards high-lipid production from volatile fatty acids (VFA) derived from anaerobic fermentation of food waste. First, we established a method for labeling intracellular lipid with lipophilic BODIPY fluorescent dye. Next, a random library was constructed by treatment with a chemical mutagen, and high-lipid producers were screened using fluorescence-activated cell sorting. Subsequently, one clone, N14, was successfully isolated, which exhibited 3-fold higher lipid production (19.4%) in VFA (6 g/L) media than the wild-type strain, and also showed increased lipid production in higher concentrations of VFA (18 or 24 g/L). Based on fatty acid methyl ester (FAME) analysis, N14 contained higher stearic acid (C18:0) and oleic acid (C18:1) content compared with those of the wild-type strain.