Invasive aspergillosis (IA) is a major concern in patients with severe immune deficiency. As antifungal susceptibility varies in different fungal pathogens, accurate and timely identification of species is becoming imperative for guidance of therapy and reducing high mortality rates in patients with IA. But, in fact, the diagnosis is challenging and new validated techniques are required for the detection and identification of clinically relevant isolates. The laser capture microdissection (LCM) system enables analysis of cytologically and/or phenotypically defined cell types from heterogeneous tissue and has been used in diagnosis and fungal species identification in pulmonary aspergillosis of white storks. To establish the experimental foundation for clinical application of the system, we microdissected and collected Blankophor-stained single hyphal strands from tissue cryosections of murine model of invasive pulmonary aspergillosis (IPA) with A. fumigatus by LCM, subsequently processed for DNA extraction, PCR sequencing, and species molecular identification. The sensitivity of LCM-PCR sequencing was 89 % (89/100), and the specificity was 100 %. Moreover, the positive predictive value and negative predictive value were 100 and 78.43 %, respectively. The result approved that the LCM-based methods had the potential for accurately diagnosis and rapidly identification fungal pathogens of IPA.