Coccidioides immitis and Coccidioides posadasii, the two recognized causes of coccidioidomycosis, may be detected by direct microscopy, culture, and serologic documentation. Two useful stains include the Grocott methenamine silver (GMS) and the calcofluor white (CFW). Other useful stains used in histopathologic studies include hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). Nucleic acid amplification tests (NAATs) have been introduced for detection of Coccidioides spp. in specimens, but are not yet commercially available. Isolation of Coccidioides spp. by culture is not difficult as many fungal as well as routine bacteriologic media are available. For the safe isolation of Coccidioides spp., the laboratory should maintain a biological safety level 2 or 3. Identification of Coccidioides spp. uses the organisms' phenotypic or genotypic characteristics. Phenotypic identification to genus level may be achieved by visualization of spherules in specimens and/or by the presence of arthroconidia in culture. Isolates may be confirmed as Coccidioides spp. by molecular probes. Separation of species into C. immitis and C. posadasii is best achieved by specialized molecular techniques which are not normally available in routine clinical laboratories. Humoral antibodies can be used for the diagnosis and prognosis of coccidioidomycosis. Although positive serologic results may be helpful in the diagnosis of coccidioidomycosis, negative serologic results cannot be used to rule out the disease. Enzyme immunoassays (EIA) and immunodiffusion methods are commonly used for detection of both IgM and IgG antibody groups. Sequential complement fixation (CF) studies for IgG class of antibody are useful for the prognosis of coccidioidomycosis.