Анотация: Real-time PCR is a sensitive and specific method for detection and differentiation of Brucella spp. This study was performed to determine the prevalence of Brucella spp. and to differentiate Brucella melitensis and Brucella abortus in cattle population in southwest Iran. A TaqMan analysis and single-step PCR was performed in total of 425 bovine blood samples. The results showed 127 (29.88 percentage) positive samples for Brucella spp. By real-time PCR 9, 69, and 5 of these specimens, were positive for B. melitensis, B. abortus, and both bacteria respectively. Results of present study indicated a high presence of this pathogen in the region. Real-time PCR is technically more simple, accurate, and rapid than current standard methods for identification and differentiation of Brucella species. To our knowledge, this study is the first prevalence report of identification and differentiation of B. abortus and B. melitensis by real-time PCR technique in Iran