Abstract Objectives The aim of this research was to set up a polymerase chain reaction (PCR)-based method to early investigate the presence of Aspergillus carbonarius in grapes. Polymerase chain reaction-based methods that target DNA are considered a good alternative with respect to traditional diagnostic methods for the early detection of pathogens because of their high specificity and sensitivity.Methods In this study, two polymerase chain reaction assays to detect the presence of A. carbonarius in grapes have been developed by using species-specific primers designed on the basis of Internal Transcribed Spacers of rDNA units and on a polyketide synthase gene responsible for ochratoxin A biosynthesis. Primers specificity was evaluated by polymerase chain reaction on A. carbonarius DNA as template and then on DNA extracted from A. carbonarius– contaminated and uncontaminated grape berries. The developed polymerase chain reaction method was used to analyze the detection limits of A. carbonarius DNA directly on grapes.Results Polymerase chain reaction amplification allows a clear detection of A. carbonarius DNA 24 hours after inoculum on grapes, i.e. when the fungus is not yet visible.Conclusion Early detection by polymerase chain reaction of the ochratoxin A producer fungus A. carbonarius could help to achieve an effective control of the fungal contamination, particularly concerning mycotoxigenic fungi.