Sigatoka disease is the most important threat for banana production worldwide. Many species of Mycosphaerella have been described from banana but, to date, the three species Mycosphaerella fijiensis, M. musicola and M. eumusae are the only species found to be pathogenic to banana. Reliable identification by classical methods requires expertise because these fungi produce similar symptoms and they are morphologically similar. For studies of ecology, genetic diversity and epidemiology their differentiation is crucial. Several laboratories have developed molecular protocols to differentiate these fungi. Currently, a number of primers targeting ribosomal sequences, actin, tubulin and histone 3 genes are available for diagnosis of the Sigatoka complex. In the present work, we report a direct colony-polymerase chain reaction (DC-PCR) approach to rapidly distinguish M. fijiensis and M. musicola strains in multiplex PCR reactions. This is the most economical and the fastest procedure reported so far for diagnosis of these two Mycosphaerella species, which are distributed in banana-growing regions in the world; the DC-PCR technique was also found to be amenable for the identification of mating type of M. fijiensis isolates. This DC-PCR may also be applicable to prepare DNA templates for basic PCR-based analyses in other fungi.