Alfalfa is believed to have originated in north-western Iran and has a long history of coexistence with its bacterial symbiont Sinorhizobium in soils of Iran. However, little is known about the diversity of Sinorhizobium strains nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations were sampled from eight different Iranian sites using three cultivars of Medicago sativa as trap host plants. A total of 982 rhizobial strains were isolated and species were identified showing a large prevalence of Sinorhizobium meliloti over Sinorhizobium medicae. Analysis of salt tolerance demonstrated a great phenotypic diversity. The genetic diversity of the Sinorhizobium isolates was analysed using BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Patterns ofBOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the role of plant variety and site of origin in the genetic variance observed. Results indicated that most of the total molecular variance was attributable to divergence among strains isolated from different sites and cultivars (intrapopulation, strain-by-strain variance). Moreover, the analysis showed the presence of two geographic populations (west and northwest), indicating that the effect of the site of origin could be more relevant in shaping population genetic diversity than the effect of cultivar or individual plant.