Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the most simple method for single-nucleotide change detection. It is widely used in the detection and differentiation between mycotoxigenic species. It is based on PCR amplification of a target region containing the variant site of the studied species followed by restriction endonuclease digestion and gel electrophoresis to visualize the RFLP patterns. In this method primers are designed to flank the polymorphic site and positioned in such a way as to create unequally sized fragments upon restriction endonuclease cleavage of the PCR products. Here, we describe the protocol of PCR-RFLP developed for the detection and differentiation between Aspergillus flavus and A. parasiticus by amplifying a 674 bp fragment of the aflR–aflJ intergenic region followed by restriction endonuclease analysis using BglII to obtain RFLP patterns.