Brucellosis is economically important infection in Egypt, and the brucellosis causative agent has not been genetically characterized in detail. In this study Brucella melitensis bv.3 was isolated and characterized from retropharyngeal lymph node and other lymphoid organs of sheep and goats from Almonofiyah Governorate & Albehera Governorate, Egypt, and to characterize one of the Br. melitensis isolates by 16S rRNA gene sequencing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR_RFLP) of outer membrane protein (omp2a) gene. Br. melitensis was isolated and identified from 17 out of 47 (36.2%) samples from lymphoid organs of sheep& goats. The 16S rRNA gene of one of the Br. melitensis isolate was sequenced and identical as Br. melitensis bv.3 strain (Gene Bank accession No. KF177277.1). The genetic characterization of Br. melitensis in this study can be used for epidemiological studies,   control of ovine brucellosis. In our knowhow this is the first report of the16S rRNA gene sequence of Brucella in Egypt.