The goal of this work was to perform a detailed analysis of fatty acids from Aspergillus species in a search for biomarkers to differentiate the spicies. In the course of this work, we isolated various classes of lipids (neutral lipids, glycolipids, phospholipids and lipopolysaccharides) from six Aspergillus species by chloroform/methanol extraction and multiple column fractionations. Further separation of structurally different fatty acids from phospholipids was also performed. The identities of isolated fatty acids from all lipid fractions were tentatively assigned using gas chromatography/mass spectrometry (GC/MS). The experimental methodology was found to be suitable for complete characterization of the fatty acids from various cellular sources. While the methods used in this work are standard techniques, the detal obtained in the fatty acid composition of each of the Aspergillus species is much greater than previously described.