Forty-eight rhizobial strains isolated from root nodules of alfalfa (Medicago sativa L.) plants growing in different regions of Iran were characterized by using restriction fragment length polymorphism (RFLP) of ribosomal internal transcribed spacer (ITS) region, 16S RNA gene-RFLP and sequence analysis of symbiotic genesnodA and nifH. Taxonomic relationships of selected strains from different ITS-RFLP groups were further inferred in this study by using single and concatenated phylogenetic analysis of recA and atpD gene sequences. In all constructed trees, the strains were distributed similarly into three different rhizobial phylogenetic groups I, II and III. Group I included strains belonging to Sinorhizobium (Ensifer) meliloti and S.kummero wiae. The strains in groupsII and III were identified as E. adhaerens and Agrobacterium radiobacter, respectively. Based on the two core genes used in this study, S.kummerowiaestrains appeared to be very closely related to S. melilotireference strains. Forty-two strains induced nitrogen-fixing nodules on alfalfa plants. Remarkably, two of these strains belonged to A. radiobacter. The entire three test strains in group II (E. adhaerens) and one test strain in group III (A. radiobacter) failed to nodulate. Generally, our results confirm that S. meliloti is the most important symbionts of M. sativa growing in agricultural fields in Iran and question S. kummerowiae being a species separate from S. meliloti.