Aspergillus flavus is an important fungal species that frequently contaminates food commodities with diverse toxins, with aflatoxins being the most relevant in food safety. In addition, this is one of the major pathogenic Aspergillus species. In this work, specific PCR-based protocol for this species is described which allows the discrimination of other closely related species from the Aspergillus section Flavi, particularly Aspergillus parasiticus. The specific primers were designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA).