Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus.