The symbiotic association between Rhizobium and leguminous species plays a significant role in sustainable agricultural systems as it contributes higher amounts of fixed nitrogen to soil through biological nitrogen fixation. Gliricidia sepium is one of the most commonly cultivated agro forestry trees in the world. This leguminous tree is widely distributed throughout Sri Lanka. A very limited number of studies are available on G. sepium-Rhizobium symbiosis in Sri Lanka. The main objective of this study is to isolate and identify the stress tolerant Rhizobium sp. in G. sepium as a preliminary approach to use rhizobial strains for cross inoculation of crop legumes in order to minimize the heavy use of chemical nitrogen fertilizers. The root nodules of G. sepium were collected from seven sites in Anuradhapura district which is located in the dry zone of Sri Lanka. A total of 34 strains were tested for their tolerance to different physiological conditions namely pH, salinity, drought and temperature. Fifteen strains showed a higher tolerance among the isolated 34 strains when grown under a wide range of physiological conditions. When these 15 stress tolerant rhizobial strains were grown under extreme physiological conditions, 12 strains could survive well as they often experience adverse environmental conditions in their natural habitat. DNA fingerprinting analysis with ERIC 1R and ERIC 2R showed that the 15 strains are genetically diverse and they belong to 9 clusters at 71% similarity level.