The diversity of bacteria nodulating Aeschynomene americana L. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK, recA, and glnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genus Bradyrhizobium, in which B. yuanmingense was the dominant species. Some of the other species, including B. japonicum, B. liaoningense, and B. canariense, were minor species. These isolated strains were divided into 2 groups-nod-containing and divergent nod-containing strains-based on Southern blot hybridization and PCR amplification of nodABC genes. The divergent nod genes could not be PCR amplified and failed to hybridize nod gene probes designed from B. japonicum USDA110, but hybridized to probes from other bradyrhizobial strains under lowstringency conditions. The grouping based on sequence similarity of nod genes was well correlated with the grouping based on that of nifH gene, in which the nod-containing and divergent nod-containing strains were obviously distinguished. The divergent nod-containing strains and photosynthetic bradyrhizobia shared close nifH sequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated from A. americana could nodulate Aeschynomene plants that belong to different cross-inoculation (CI) groups, including A. afraspera and A. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic and nod-containing strain) originating from CI group 1 nodulating roots of A. indica (CI group 3). An infection process used to establish symbiosis on Aeschynomene different from the classical one is proposed. © 2012, American Society for Microbiology. All Rights Reserved.