Fungal infections are increasing, particularly among immunocompromised hosts, and a rapid diagnosis is essential to initiate antifungal therapy. Often fungi cannot be identified by conventional methods and are classified as nonsporulating molds (NSM).We sequenced internal transcribed spacer regions from 50 cultures of NSM and found 16 potential pathogens that can be associated with clinical disease. In selected clinical settings, identification of NSM could prove valuable and have an immediate impact on patient management.Fungal infections are increasing, particularly among immunocompromised hosts, and a rapid, accurate diagnosis is essential for the initiation of targeted antifungal therapy. Diagnosis of fungal infections usually depends on recovery of fungi from culture of clinical specimens, and their identification requires the presence of reproductive structures. Often fungi cannot be characterized fully because the mold does not sporulate, making identification by microscopic morphology not possible and potentially increasing the time to report an inconclusive result to 21 days. While many laboratorians and clinicians assume that these fungal isolates are environmental organisms and not clinically significant, to our knowledge no study has systematically attempted to classify these previously unidentifiable fungi in a clinical microbiology laboratory.Amplification and sequencing of target regions within the ribosomal DNA gene complex has emerged as a useful, adjunctive tool for the identification of fungi and does not depend on mold sporulation for identification (3,5,9,11). The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (2,3,5,9,11,15). The goals of this study were to determine if sequencing the ITS 1 and 2 regions of nonsporulating molds (NSM) could identify fungi that were not identifiable by conventional methods and could serve as an approach to detect clinically relevant pathogens.Sample selection. Identification of molds directly from a specimen or submitted as an isolated culture to Associated Regional and University Pathologists, Inc., Laboratories (ARUP Laboratories) was attempted by growth on inhibitory mold agar, modified Sabouraud agars, or potato dextrose agar. Microscopic structures were observed on tease or tape preparations and slide cultures for up to 21 days. NSM were defined as molds without reproductive structures and that could not be further characterized.