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The diagnosis of histoplasmosis is based on a multifaceted approach that includes clinical, radiographic, and laboratory evidence of disease. The gold standards for laboratory diagnosis include demonstration of yeast on pathological examination of tissue and isolation of the mold in the culture of clinical specimens; however, antigen detection has provided a rapid, noninvasive, and highly sensitive method for diagnosis and is a useful marker of treatment response. Molecular methods with improved sensitivity on clinical specimens are being developed but are not yet ready for widespread clinical use. This review synthesizes currently available laboratory diagnostics for histoplasmosis, with an emphasis on complexities of testing and performance in various clinical contexts.