Although histopathology is required for definitive diagnosis of fungal infections, conclusive identification and discrimination of fungi in tissue sections and cytological preparations remain technically difficult. Therefore, new diagnostic tools are needed for the routine diagnosis of pathogenic fungi. In situ hybridization (ISH) is a non-culture based procedure that has many advantages over traditional diagnostics for identification of pathogenic fungi in histological specimens. This review highlights the basic ISH technique, with particular emphasis on using pretreatment of tissue sections prior to hybridization to solve problems associated with formalin fixation. With this modification, ISH has become a valuable tool that complements conventional histopathological diagnoses in formalin-fixed and paraffinembedded (FFPE) tissues. However, understanding the limitations imposed by formalin fixation is essential in developing suitable ISH protocols for fungal identification.