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ABSTRACTDiagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillusinfections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillusspecies (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillusand species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatusin untreated (P< 0.0007) and treated (P= 0.008) animals, respectively. For infections caused by A. terreusand A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-AspergillusPCR was more sensitive than culture for detection of A. flavusin treated animals (P= 0.002). BAL fluid pan-Aspergillusand species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillusand species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.