PCR can aid in the diagnosis of invasive fungal disease (IFD). While the large number of "in-house" methodologies drives technological diversity, providing robustness, they make it difficult to identify optimal strategies, limiting standardization, and widespread acceptance. No matter how efficient, PCR utility will be limited by the quality of extracted nucleic acid. This chapter highlights benefits and limitations affecting the nucleic acid extraction process, before focusing on recent recommendations that through multicenter evaluation have provided optimal and standardized methodology.