Rhodotorula glutinis is an oleaginous yeast that has the ability to produce up to 70% of its weight as oil in the form of triacylglycerol (TAG). The potential to use this oil as a feedstock of biodiesel production is considerable. The ulterior purpose is to use glycerol as a growth substrate for the yeast either solely or in combination with glucose or xylose. Glycerol is a byproduct of transesterification reaction of TAG. When TAG is reacted with an alcohol such as methanol in the presence of a catalyst, the ester bonds are broken and the fatty acids are methylated leaving the glycerol backbone as a by-product. The result is 90% fatty acid methyl esters (FAMEs) and 10% glycerol. Biodiesel is a source of energy that is a mixture FAMEs. Therefore, the generation of FAMEs for the manufacturing of biodiesel produces considerable amounts of glycerol that, when recycled back into the production system, would provide an inexpensive carbon source for which to grow oleaginous yeast R. glutinis. This study is designed to determine the feasibility of the oleaginous yeast R. glutinis as a producer of FAMEs since it accumulates TAG when grown on carbon substrates such as xylose, glucose, and glycerol. Preliminary results indicate that R. glutinis grown on glycerol and glucose as carbon substrates produce up to 31% dry cell weight (w/w) of TAG after 48 h. When grown on glycerol and xylose, the organism produced 17% TAG (w/w). From the results obtained, we can recommend that R. glutinis is a viable source of TAG for the production of biodiesel and that glycerol can be used in combination with glucose or xylose as a growth substrate to produce TAG. Future research will focus of the optimization of the conditions for yield TAG improvement.