Polymerase chain reaction–restriction fragment length polymorphism markers were developed to study populations of the fungal banana pathogen Mycosphaerella fijiensis. Twelve markers were defined, 11 in anonymous and single-copy nuclear DNA sequences and one in the internal transcribed spacer and 5.8S rDNA sequence. The polymerase chain reaction products obtained with locus-specific primer pairs were digested with restriction enzymes to reveal polymorphism. Between five and 12 markers were polymorph in M. fijiensis populations from different geographical origins (Papua New Guinea, the Philippines, Cameroon and Latin America). The mean of allele number and gene diversity (expected heterozygosity) per locus in the different geographical populations ranged between 1.4 and 2.7 and 0.17 and 0.45, respectively.