Brucellosis is one of the zoonotic diseases of major concern and can cause huge economic losses to livestock industry. Serological tests and bacterial isolation are considered as the gold standard assays for diagnosis of Brucella spp. but they are time-consuming, hazardous and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore, in this study three real time TaqMan PCR assay to detect (IS711 gene based) and differentiate B. abortus (BruAB_0168 gene) and B. melitensis (BMEII0466 gene) were designed and evaluated in singlex and multiplex formats.