Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a <1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a <1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.Aspergillus species are an increasingly important cause of invasive fungal infections in immunocompromised patients (31, 59). Unfortunately, there are few specific clinical signs of invasive aspergillosis and current methods for laboratory diagnosis are less than ideal, particularly in the early stages of the disease (8,49). Given the recent reports of reduced antifungal drug susceptibilities among some Aspergillus species (21, 26, 50), the timely and accurate identification of aspergilli to the species level has become especially important (10). Species identification is also important for epidemiological purposes and as a guide to clinical management (29,47,48).The current laboratory identification of Aspergillus species is based on macroscopic colonial and microscopic morphological characteristics (7,20,45). Over 180 different species in at least 16 subgeneric groups or sections can be distinguished (35,37,38), including approximately 30 species which are recognized as opportunistic pathogens of humans (7). Many clinical laboratories use traditional phenotypic methods of identification and can differentiate only the more common Aspergillus species; the delineation of less common species must b...