The symbiotic relationship between host plant – alfalfa (Medicago sativa L.) and nodule bacteria Sinorhizobium meliloti is considered to be very significant in sustainable agricultural production. Symbiotic nitrogen fixation provides input of significant nitrogen amounts in agricultural soil, which enable reduced application of mineral nitrogen fertilizers. The realization of these relations is possible by legumes seed inoculation, where are highly effective strains of rhizobia, with well known characteristics, used as an inoculum. Except of presence of some other nitrogen sources, the bacterization efficacy can be reduced also by presence of competitive indigenous rhizobial strains in soil. Such strains can be adapted to the all conditions in soil, infect the present host plant, but it is not necessarily that the process of nitrogen fixation will happen, or that sufficient quantity of nitrogen will be fixed. That is why the isolation and characterization of indigenous strains of S. meliloti is so important, and that was also the objective of this study. Indigenous strains were isolated from the soil collected in the Istrian, Zadar, Koprivnica – Križevci and Brod – Posavina county. The study includes 20 indigenous strains, one reference strain S. meliloti 2011 and one standard strain Sinorhizobium medicae 18864. Identification to the species level was performed by 16S rDNA PCR – RFLP method, and after that isolates which were not S. meliloti species were reduced. It has been found that 14 of 20 indigenous strains belongs to the specified species, while the other 6 were significantly different. Identification to the strain level was performed by RAPD – PCR and ERIC – PCR methods. Through identifications of these isolates done by specified methods, significant genetic variability was established within natural populations of S. meliloti. Further fenotipic and simbiotic characterizations of this strains are required in order to select the strains with highest qualities for potential use in alfalfa cultivation.