A hierarchical collection of isolates of the fungal pathogen Pleiochaeta setosa was made from field-grown Lupinus albus plants across southern NSW, Australia in 2004. This collection, along with some historical reference isolates, was analysed for genetic variation using random amplified polymorphic DNA and enterobacterial repetitive intergenic consensus primer PCR. Anchored inter-simple sequence repeat primers were tested but produced little or no amplification or no detectable polymorphism. The observed overall variation was not explained by the hierarchical structure of the population (individual seedling, host species, host cultivar, regional location, or year). There was significant genetic variation between isolates even at the finest sampling scale (within a single seedling). Some very distinct individual isolates were identified. The high level of scale-independent genetic diversity seen in P. setosa suggests that field-based screening for Pleiochaeta root rot-resistant genotypes in a L. albus breeding program at a single location is likely to result in resistance which is effective at multiple and widely separated locations. This resistance would have an increased chance of being polygenically controlled and durable under commercial cultivation conditions.