Premise of the study: Large-scale population genetics studies are required to investigate the dispersal processes underlying the emergence of Mycosphaerella fi jiensis , a fungal pathogen of banana. To this end, we have developed an optimized genotyping procedure combining novel microsatellite markers and a modifi ed DNA extraction protocol. Methods and Results: Primers for tetranucleotide loci were designed directly from the recently published genome sequence of M. fijiensis. A total of 19 new polymorphic and easy-to-score markers were developed. Their use was combined with an adapted protocol for total DNA extraction starting from young lesions collected from banana leaves, thus avoiding a pathogen isolation step. Conclusions: The combination of the two technical developments presented here will permit the expansion of genotyping capacity in M. fijiensis, allowing large-scale analysis of samples from various geographic locations.