This invention, entailsnucleic acid-based assays, for detecting the presence of pathogenic fungi such as Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Pneumocystis brasiliensis, and/or Penicillium marneffei within a sample. Within a healthcare setting, this particular approach can greatly reduce pathogen identification time, better direct treatments and ultimately improve patient outcomes. Further, this technology provides improved diagnostic specificity compared to serologic tests for circulating antibodies using patient serum samples- an approach that may give particularly aberrant results for immunosuppressed individuals, and who are frequently afflicted with opportunistic fungi. This technology is readily adaptable as kits used for species-specific identification of fungal pathogen infections and environmental contamination.