Sunflower (Helianthus annuus) is host to infections by Sclerotinia sclerotiorum originating from either homothallic sexually-derived ascospores (stem and head rots) or asexually-derived sclerotia (root rot). While sunflower can be infected by either ascospores or sclerotia this study found no association between the genotypes found in lesions and the type of infection (stem, head or root rot). Multicopy Restriction Fragment Length Polymorphisms (RFLPs) showed individual sclerotia comprised of only one genotype, and that all eight ascospores within an ascus also had only one genotype. Mycelial Compatibility Groups (MCGs), Random Amplified Polymorphic DNAs (RAPDs), single and multicopy RFLP analyses all showed the majority of sunflower plants were infected by only one genotype. A sample of 250 isolates collected hierarchically from sunflowers in Queensland and New South Wales were shown to belong to one large genetic population of S. sclerotiorum. Temporal studies revealed genetic uniformity was maintained across years, further confirming one genetic population. A range of molecular markers were used to genotype 120 isolates, resulting in differing levels of resolution of a genotype. Between 13 and 24 genotypes were identified with similarities and differences in the assemblages of isolates within each genotype depending on the marker used.