The incidence of invasive fungal infections (IFI) has increased substantially and the epidemiology has changed dramatically in recent years. Candida albicans is still most important, but non-albicans species, Aspergillus species, Glomeromycota (formerly Zygomycetes) and Fusarium species are an increasing cause of IFIs. Due to this growing diversity, the identification of the causative organism to genus and species level is important to perform best and adequate treatment. The early, sensitive and specific detection of IFIs remains challenging and current conventional methods are limited. The golden standard for the definite diagnosis of proven pulmonary infection remains either histopathologic, cytopathologic or direct tissue examination. Invasive procedures are necessary to obtain reliable specimens and biopsies may be taken percutaneously, bronchoscopically, via open surgery or via video-assisted thorascopic surgery. Molecular methods, like PCR or in situ hybridization, are a promising diagnostic tool for rapid and reliable species identification and should be performed in addition to microscopic examination and culture to increase the sensitivity for the diagnosis of IFI. Combining culture, microscopy, serology, and PCR in lung tissues and/or bronchial samples will increase the diagnostic yield by 99%. Here, we give an overview of biopsy procedures for molecular tissue diagnosis of IFI.