Grass pea (Lathyrus sativus) is an insurance crop grows extensively in the world and has symbiosis interaction with nitrogen fixing rhizobia. Farmers used glyphosate as weed control in agricultural process. However, the application this chemical herbicide affects soil fertility by decreasing nitrogen fixing rhizobia. The objective of this study was to assess the effects of glyphosate on the Rhizobium isolate. Grass pea with good growth and healthy appearance were selected purposefully form the field and rhizobia was isolated from root nodules. Isolates were characterized on the basis of their morphological, biochemical characteristics and resistance of abiotic condition. Isolates of rhizobia from grass pea were gram negative; circular, milky, 2-4.1mm diameter and most of them are smooth colony appearance on YEMA. Based on the morphological and biochemical characteristics of isolates were grouped under the genus Rhizobium. Isolates was grown best at the temperature between 28-38 , pH 5-8 and at 0.5 and 1% salt concentration. Glyphosate was found to affect the survival of Rhizobium under laboratory. 5.52-47% of Rhizobium population was inhibited at 20ml L concentration of glyphosate. At the concentration of 40mlL -1 percentage of inhibition was 17.1% - 53.38%. However, 87% of isolates were inhibited with higher concentration (60ml L −1 ) of glyphosate. The number of colonies after the exposure of glyphosate was greatly dependent on concentration. Thus isolate from root nodules of grass pea was inhibited by glyphosate under laboratory condition. Future work needs to identify resistant Rhizobium from grass pea root nodule through evaluating their responses under greenhouse and field conditions and used as microbial inoculum.