In the last few decades, there has been a growing interest in environmentally friendly sustainable agricultural practices, thus increasing the role of biofertilizers such as rhizobia, which can decrease the need for chemical fertilizers, reduce adverse environmental effects, and help to save money. Therefore, information on the distribution and genetic variation of native rhizobial isolates would aid in selecting novel rhizobial strains that could be developed and used as biofertilizers in legume production. This research was conducted to characterize 24 rhizobial isolates from five legumes on morphological, biochemical, and molecular aspects and determine the phylogenetic relationships among them. Rhizobial isolates were obtained from five Egyptian legumes: faba bean, lentil, pea, clover, and soybean. Morphological characterization classified the isolates into fast and slow growers. Biochemical characterization using API 20E and API 20NE systems showed a large diversity, which may reflect their adaptation in different environments. Moreover, molecular detection of the 16S rRNA gene enabled to characterize 19 of them to the species level. Rhizobial isolates from pea, faba bean, clover, and lentil were identified as Rhizobium leguminosarum and those from soybean were identified as Bradyrhizobium japonicum. These data reflected a narrow diversity of rhizobial species in Egypt. A phylogenetic analysis of the 19 isolates confirmed that B. japonicum isolates were divergent from all other isolates. Furthermore, the phylogram revealed that each group of isolates that originated from the root nodule of a certain legume formed a separate subcluster. The obtained data suggested a narrow range of interspecies variations, which is consistent with the idea of the presence of biovars among the species.