ABSTRACT Aspergillus fumigatus has been understood to be the most common cause of invasive aspergillosis (IA) in all epidemiological surveys. However, recent studies have uncovered a large degree of genetic heterogeneity between isolates morphologically identified as A. fumigatus , leading to the description of a new species, Aspergillus lentulus . Here, we examined the genetic diversity of clinical isolates identified as A. fumigatus using restriction enzyme polymorphism analysis and sequence-based identification. Analysis of 50 clinical isolates from geographically diverse locations recorded the presence of at least three distinct species: A. lentulus , Aspergillus udagawae , and A. fumigatus . In vitro, A. lentulus isolates demonstrated decreased susceptibility to antifungal drugs currently used for IA, including amphotericin B, voriconazole, and caspofungin; A. udagawae isolates demonstrated decreased in vitro susceptibility to amphotericin B. Results of the present study demonstrate that current phenotypic methods to identify fungi do not differentiate between genetically distinct species in the A. fumigatus group. Differential antifungal susceptibilities of these species may account for some of the reported poor outcomes of therapy in clinical studies.