Penicillium species have been studied for over 200 years and the genus was first described by Link in 1809. Initially, morphological identification methods were used however, much diversity within the genus resulted in researchers seeking alternative techniques and approaches to improve accuracy. These methods involved biochemical analysis of secondary metabolites in conjunction with morphological examination. With the emergence of more accurate and rapid molecular identification tools, scientists embraced modem technology to address diversity challenges. In order to provide a more holistic approach towards the taxonomy of complex genera, morphological analysis remains an essential component in Penicillium identification. Penicillium species are omnipresent, dominant and problematic in postharvest environments. They are known to cause major losses in export markets due to fruit decay. The aim of this study was to identify species within the South African litchi export chain and develop a rapid method for Penicillium identification. This study used morphological as well as molecular identification methods in order to develop PCR-RFLP restriction maps for a number of dominant Penicillium species. Seventeen species of Penicillium were identified using conventional morphological methodology and DNA sequencing, both of which are laborious and time-consuming. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism provided reliability and repeatability as well as being a cost-effective and rapid identification alternative. A combined phylogenetic study indicated that the taxonomic position of several species may need to be reconsidered. Fourteen species were differentiated from one another through digestion of the â-tubulin gene region with five restriction enzymes. Banding patterns correlated well with phylogenetic and biochemical data of related studies, indicating that this method holds promise as a rapid identification procedure for Penicillium species.