Symbiotic relationships between legumes and nitrogen-fixing soil micro-organisms are of ecological importance in plant communities worldwide. For example, nutrient-poor Australian soils are often dominated by shrubby legumes (e.g. species of Acacia). However, relatively few studies have quantified patterns of diversity, host-specificity and effectiveness of these ecologically important plant-microbe interactions. In this study, 16S rRNA gene sequence and PCR-RFLP analyses were used to examine bacterial strains isolated from the root nodules of two widespread south-eastern Australian legumes, Acacia salicina and Acacia stenophylla, across nearly 60 sites. The results showed that there was extensive genetic diversity in microbial populations, including a broad range of novel genomic species. While previous studies have suggested that most native Australian legumes nodulate primarily with species of the genus Bradyrhizobium, our results indicate significant associations with members of other root-nodule-forming bacterial genera, including Rhizobium, Ensifer, Mesorhizobium, Burkholderia, Phyllobacterium and Devosia. Genetic analyses also revealed a diverse suite of non-nodulating bacterial endophytes, only a subset of which have been previously recorded. Although the ecological roles of these endosymbionts are not well understood, they may play both direct and indirect roles in promoting plant growth, nodulation and disease suppression.