ABSTRACT Acrophialophora fusispora is an emerging opportunistic fungus capable of causing human infections. The taxonomy of the genus is not yet resolved and, in order to facilitate identification of clinical specimens, we have studied a set of clinical and environmental Acrophialophora isolates by morphological and molecular analyses. This set included the available type strains of Acrophialophora species and similar fungi, some of which were considered by various authors to be synonyms of A. fusispora . Sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA and a fragment of the β-tubulin ( Tub ) gene revealed that Acrophialophora belongs in the family Chaetomiaceae and comprises three different species, i.e., A. fusispora , Acrophialophora levis , and Acrophialophora seudatica ; the latter was previously included in the genus Ampullifera . The most prevalent species among clinical isolates was A. levis (72.7%), followed by A. fusispora (27.3%), both of which were isolated mostly from respiratory specimens (72.7%), as well as subcutaneous and corneal tissue samples. In general, of the eight antifungal drugs tested, voriconazole had the greatest in vitro activity, while all other agents showed poor in vitro activity against these fungi.