Previous research reveals that the genome structures of rhizobial type strains and reference strains can reflect their phylogenetic relationships. In order to further explore the potential application of genome structure as a phylogenetic marker in rhizobial natural taxonomy, this study analyzed the genome structures of 29 unclassified nodule bacteria isolated from the root nodules of leguminous trees, Robinia sp., Dalbergia spp., and A lbizia spp. and 7 rhizobial reference strains by I-CeuI cleavage, then clustered these bacteria phylogenetically based on their genome structures and compared these clusters with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Eleven phylogenetic clusters were obtained. The clusters were in large part consistent with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Also there are inconsistent clusters based on the above three methods. But results are completely consistent with 16S rRNA clusters. This suggested that the genome structure clustering method can be used to fastly identify root nodule isolates and detect their phylogenetic relationships. The credibility and repeatability of the results, together with the simplicity and possibility to analyze a large number of strains in a short time of the method, indicates the broad potential application of genome structure as phylogenetic marker to categorize rhizobial isolates and should in the future facilitate biodiversity studies.