A study on development of a rapid PCR-based detection method based on ITS region of M. eumusae, M. Fijiensis, and M. musicola on banana was carried out. The main objecive of this study was to develop a fast and species-specific PCR-based detection method for the presence of Mycosphaerella species on banana. The methods include collection of specimens, morphological identification supported by molecular phylogenetic analysis, RFLP analysis, species-specific primers development, and validation. Two species of Mycosphaerella, namely, M. Fijiensis and M. musicola, and one unidentified Pseudocercospora species were found in Java island. Three restriction enzymes used in the RFLP analysis, viz, AluI, HaeIII, and TaqI were capable to discriminate M. eumusae, M. Fijiensis, and M. musicola. Two species-specific primer pairs, viz, MfijF/MfijR and MmusF/MmusR have been successfully developed to detect for the presence of M. Fijiensis and M. musicola, respectively.