Mycotoxins are secondary metabolites produced by filamentous fungi which have toxicologically relevant effects on vertebrates when administered in small doses via a natural route. In order to improve food safety and to protect consumers from harmful contaminants, presence of fungi with the potential to produce such compounds must be checked at critical control points during production of agricultural commodities as well as during the process of food and feed preparation. Polymerase chain reaction (PCR) based diagnosis has been applied as an alternative assay replacing cumbersome and time consuming microbiological and chemical methods for detection and identification of the most serious toxin producers in the fungal genera Fusarium, Aspergillus, and Penicillium. The current review covers the numerous PCR-based assays which have been published over the last decade since the first description of the use of this technology to detect aflatoxin biosynthesis genes in A. flavus.