Phragmidium violaceum causes leaf rust on the European blackberry (Rubus fruticosus L. aggregate). Multiple strains of this pathogen have been introduced into southern Australia for the biological control of at least 15 taxa of European blackberry, a nonindigenous, invasive plant. In climates conducive to leaf rust, the intensity of disease varies within and among infestations of the genetically variable host. Genetic markers developed from the selective amplification of microsatellite polymorphic loci were used to assess the population genetic structure and reproductive biology of P. violaceum within and among four geographically isolated and diseased infestations of the European blackberry in Victoria, Australia. Despite the potential for long-distance aerial dispersal of urediniospores, there was significant genetic differentiation among all populations, which was not associated with geographic separation. An assessment of multilocus linkage disequilibrium revealed temporal and geographic variation in the occurrence of random mating among the four populations. The presence of sexual spore states and the results of genetic analyses indicated that recombination, and potentially random migration and genetic drift, played an important role in maintaining genotypic variation within populations. Recombination and genetic differentiation in P. violaceum, as well as the potential for metapopulation structure, suggest the need to release additional, genetically diverse strains of the biocontrol agent at numerous sites across the distribution of the Australian blackberry infestation for maximum establishment and persistence.