Illinois bundleflower [Desmanthus illinoensis (Michx.) MacMillan] is an herbaceous perennial legume native to North America. Useful as a N2-fixing plant in warm-season grass pastures, Illinois bundleflower is also a promising perennial grain crop. Knowledge of the distribution of genetic variation in Illinois bundleflower would increase efficiency of germplasm preservation and expedite plant breeding progress. The objective of this experiment was to determine the distribution of genetic variation within and between Illinois bundleflower accessions by amplified fragment length polymorphism (AFLP) markers. Semi-automated fluorescence-based AFLP analysis was performed on three individuals from each of 50 accessions. We identified 222 markers, 159 of which were polymorphic. Within-accession diversity was low (H(S) = 0.013) compared with total gene diversity (H(T) = 0.086). Analysis of molecular variance (AMOVA) showed that 83% of the molecular variance was explained by two major clusters, supporting a previous phenotypic study that suggested the existence of two distinct races of Illinois bundleflower. The less commonly occurring of the two races contained greater genetic diversity and originated from a localized region in the south central USA. The collection of more accessions from throughout the USA and focused on the Southeast would likely increase the genetic variation available to plant breeders.