Aims Colletotrichum kahawae is a strongly aggressive pathogen causing coffee berry disease and is specific to Arabica coffee (Coffea arabica) in Africa. In this article, we developed a real-time PCR assay for the species-specific diagnosis of C.kahawae by designing the primers and a TaqMan probe derived from the single nucleotide polymorphism-rich region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Methods and Results DNA markers from rDNA internal transcribed spacer, actin, -tubulin and GAPDH genes of the ex-type culture of C.kahawae and 10 reference strains of Colletotrichum species were analysed for intra- and interspecific variations. The GAPDH gene was selected to develop a species-specific DNA marker. A TaqMan real-time PCR assay for species-specific detection of C.kahawae was developed, and its accuracy was tested against type strains of other phylogenetically closely related species in the C.gloeosporioides species complex, with the detection sensitivity of 80fgl1 of genomic DNA. Conclusions This real-time PCR assay is highly specific and sensitive for the diagnosis of C.kahawae and can be applied in qualitative and quantitative tests. Significance and Impact of the Study This protocol allows for a rapid and sensitive detection of C.kahawae and will be useful in disease management and pest detection to prevent further spread of this pathogen.