The diagnosis of blastomycosis and histoplasmosis can be difficult for clinicians who rarely see infections caused by these environmentally restricted dimorphic fungi. Historically, the diagnosis of blastomycosis has been established by culture and sometimes by histopathologic identification. Currently, antigen detection in urine and serum has been shown to aid in the rapid diagnosis of blastomycosis, and newer antibody assays are likely to contribute to our diagnostic capability in the near future. The gold standard for the diagnosis of histoplasmosis has been culture of the organism from involved tissues, aided in some patients by histopathological verification of the typical yeast forms in tissues. Antigen detection has contributed greatly to the ability of clinicians to rapidly establish the diagnosis of histoplasmosis, especially in severely ill and immunocompromised patients, and antibody testing for provides important adjunctive diagnostic capability for several forms of both acute and chronic histoplasmosis. For both of these endemic mycoses, novel molecular tests are under active investigation, but remain available in only a few reference laboratories. In this review, we provide a synopsis of diagnostic test options that aid in establishing whether a patient has blastomycosis or histoplasmosis.