Species of the Aspergillus niger aggregate are known to produce feruloyl esterases, enzymes involved in the degradation of cell wall polymers. However, species delineation is difficult in these fungi. We combined AFLP analysis with ITS rDNA and beta-tubulin sequencing to characterize the isolates of this aggregate in terms of feruloyl esterase production. A preliminary re-examination of isolates based on comparison of ITS rDNA and beta-tubulin sequences with those of typical taxa deposited in international collections led us to re-identify the isolates as members of the species A. niger, A. foetidus and A. tubingensis. Molecular clustering based on beta-tubulin data and AFLP analysis showed that the strains of A. niger formed a homogenous phylogenetic group distinguished by either zero or type A feruloyl esterase activity, while strains A. foetidus and A. tubingensis exhibited type B feruloyl esterase activity when grown on sugar beet pulp.