Numerous thrips species cause economic damage to a range of agricultural crops in southern Africa. Much of this economic damage may be prevented by an accurate system for identifying pest thrips species. At present the inability to identify thrips species leads both to poor control in the field and a decrease in exports due to the presence of species of quarantine importance. The objective of this study was therefore to provide a thrips identification system for a selection of economically important species present in southern Africa. For this purpose, a molecular identification tool, based on nucleotide sequencing analysis of the mitochondrial cytochrome oxidase I (COI) gene, was developed. Nine thrips species were included in analyses: Frankliniella occidentalis, F. schultzei, Fulmekiola serrata, Haplothrips nigricornis, H. clarisetis, Heliothrips sylvanus, Scirtothrips aurantii, Synaptorthrips psoraleae and Thrips tabaci. Analysis of 163 individuals showed that each of the nine thrips species could be distinguished on the basis of their COI sequences. Information generated was integrated with that produced previously for thrips species present in Europe and allowed a rapid, accurate and simple means of identifying the species of major economic importance present in southern Africa. The molecular key will provide much-needed information on thrips identification for South African pest management officers and quarantine purposes.