A microsatellite-based DNA profiling system was developed that can be used to distinguish genetically diverse isolates of a forest pathogen, Dothistroma pini. All isolates of this pathogen identified in New Zealand so far appear to be isogenic and the disease is kept under control by aerial applications of copper fungicide. Although New Zealand has strict importation controls in place, the possibility of genetically diverse isolates of D. pini being introduced from other countries poses a severe biosecurity threat to forest health. Therefore a DNA-based monitoring system was developed. Two informative microsatellite loci were found serendipitously in D. pini sequence data available in our laboratory. Further microsatellite loci were obtained using a rapid 5′-anchored PCR amplification technique. For each informative locus identified, specific primers were designed to flank the repeated sequence and subsequently used to generate DNA profiles for the D. pini strains. The profiles obtained from five microsatellite loci were sufficient to distinguish most isolates tested. The anchored primer technique provides an efficient tool for the identification of polymorphic loci that can be used to screen for genetic differences between fungi.