BACKGROUND: Symbiotic relationships between leguminous plants (family Fabaceae) and nodule-forming bacteria in Australia native ecosystems remain poorly characterized despite their importance. Most studies have focused on temperate parts of the country, where the use of molecular approaches have already revealed the presence of Bradyrhizobium, Ensifer (formerly Sinorhizobium), Mesorhizobium and Rhizobium genera of legume root-nodule bacteria. We here provide the first molecular characterization of nodulating bacteria from tropical Australia. METHODOLOGY/PRINCIPAL FINDINGS: 45 nodule-forming bacterial strains, isolated from eight native legume hosts at eight locations in Kakadu National Park, Northern Territory, Australia, were examined for their genetic diversity and phylogenetic position. Using SSU rDNA PCR-RFLPs and phylogenetic analyses, our survey identified nine genospecies, two of which, Bradyrhizobium genospp. B and P, had been previously identified in south-eastern Australia and one, Mesorhizobium genospecies AA, in southern France. Three of the five newly characterized Bradyrhizobium genospecies were more closely related to B. japonicum USDA110, whereas the other two belonged to the B. elkanii group. All five were each more closely related to strains sampled in various tropical areas outside Australia than to strains known to occur in Australia. We also characterized an entirely novel nodule-forming lineage, phylogenetically distant from any previously described rhizobial and non-rhizobial legume-nodulating lineage within the Rhizobiales. CONCLUSIONS/SIGNIFICANCE: Overall, the present results support the hypothesis of tropical areas being centres of biodiversity and diversification for legume root-nodule bacteria and confirm the widespread occurrence of Bradyrhizobium genosp. B in continental Australia.