Previous studies have suggested that genomes A and B are two of the three genomes of the hexaploid species Stylosanthes erecta. To identify the third constituent genome of S. erecta, STS (sequence-tagged-site) and RFLP (restriction fragment length polymorphism) markers were used to analyse genotypes representing several taxa that had not been tested previously. It was found in this study that 86% of S. erecta fragments of neither A or B genomes were found in S. angustifolia, and that S. angustifolia contributed at least one of its fragments to marker profiles of S. erecta for all of the markers tested. Data from a chloroplast DNA clone suggested that S. angustifolia and S. erecta have different cytoplasm. The molecular evidence is consistent with the notion that S. angustifolia is the likely third putative diploid progenitor of S. erecta, with Genome A as the cytoplasm donor of the hexaploid species. These findings are discussed in the context of the morphology and geographical distribution of the species concerned.