The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.