This invention relates to assays for the detection and species-specific identification of Aspergillus fungi. Accurate clinical diagnosis of Aspergillus species has become increasingly important as certain species, such as A. terreus and A. fumigatus, are resistant to specific commonly employed antifungal compounds. Most contemporary fungal diagnostic methods are time-consuming and inaccurate. This invention directly addresses those inadequacies by providing a method to rapidly and accurately differentiate all medically important species of Aspergillus based on differences in the DNA sequences of the internal transcribed spacer 1 region of ribosomal DNA.