A total of ten Ensifermeliloti were isolated from root nodules of Medicago sativa L. (alfalfa) collected from different areas of Babylon province of iraq through the period of November-2012 to February- 2013. The isolates were identified using polymerase chain reaction (PCR) technique, in addition to traditional methods.The PCR analysis showed that the isolates were harboured the nodbox 4 locus and the mucR genes which confirm that these isolates belong to E. meliloti.The symbiotic properties of the isolates atpH 5 - 10 were studied. It was found that the optimum pH for growing of Em1, Em3, Em4, Em6 and Em8 isolates was pH7 , while for the remaining isolates, represented by Em2, Em5, Em7, Em9 and Em10was pH8. All isolates were able to infect the root hairs and form nodules on the roots of alfalfa plants (Nod + )at pH7,pH7.5 and pH8. The plants nodulated by these isolates were healthy and green (indicating that nitrogen was being fixed). However , the growth of isolates at pH6.5 (except for Em1 and Em8 isolates )and pH8.5 were unable to infect the root hairs and failed to elicite nodules on M. sativa (Nod - ).These plants were weak, stunted and became chlorotic six weeks after inoculation and resembled to the control (without inoculum) plants in all respects. These results indicate that the pH was significant factor and play essential role in the optimum nitrogen fixation .