Acacia gummifera is an important tree from the south-west of Morocco. It has been reported to increase the soil fertility as the other Acacia. The characterization of successful strains and the study of the potential for fixing atmospheric nitrogen was a necessary in order to succeed its establishment in the environment where it will be introduced. Growth in YMA, tolerance to stress factor (pH, salt and temperature) and resistance to metallic ions were used as phenotypic markers of isolates collected from root nodule of A. gummifera. Genotypic diversity was studied by amplification of polymorphic DNA and 16s RNA gene sequencing. The symbiosis effectiveness of 6 performed Rhizobium was evaluated using plant nodulation assay at two different ages in controlled condition.