Single-copy RFLP markers were developed for the root endophytic fungus Phialocephala fortinii. After an initial screening of 40 probes with four restriction enzymes, EcoRV and HindIII were chosen to analyse 31 strains of P. fortinii and nine strains of a closely related morphotype (Type 1) which have known ISSR-PCR or allozyme phenotypes. Type 1 isolates and a single genetically different isolate with Type 1 like morphology, a representative of a possible third taxon, showed unique alleles with several probes and both restriction enzymes. Consequently, both taxa were easily distinguishable from P. fortinii isolates. Whereas EcoRV in combination with seven probes revealed only eight multi-locus haplotypes among 17 ISSR phenotypes, HindIII in combination with 12 probes was able to distinguish all ISSR and/or allozyme phenotypes except two pairs of strains. Strains of P. fortinii showed a high gene diversity, and up to 15 alleles per locus were observed among the 31 strains of P. fortinii. The 12 probes used in combination with HindIII were (partially) sequenced and BLAST searches showed no similarities with known sequences indicating that they probably are non-coding regions of the genome. Enzyme-probe combinations suitable to indicate the genetic structure of P. fortinii and Type 1 populations were easily identified in the present study. This opens up avenues to study communities of the P. fortinii complex exposed to various environments and management intensities, and will help to promote the understanding of these extremely successful organisms.