<sec><title>Objectives</title><p>Brucellosis is a major zoonotic disease that poses a significant public health threat worldwide. The classical bacteriological detection process used to identify <italic>Brucella</italic> spp. is difficult and time-consuming. This study aimed to develop a novel molecular assay for detecting brucellosis.</p></sec><sec><title>Methods</title><p>All complete sequences of chromosome 1 with 2.1-Mbp lengths were compared among all available <italic>Brucella</italic> sequences. A unique repeat sequence (URS) locus on chromosome 1 could differentiate <italic>Brucella abortus</italic> from <italic>Brucella melitensis</italic>. A primer set was designed to flank the unique locus. A total of 136 lymph nodes and blood samples were evaluated and classified by the URS-polymerase chain reaction (PCR) method in 2013–2014.</p></sec><sec><title>Results</title><p>Biochemical tests and bacteriophage typing as the golden standard indicated that all <italic>Brucella</italic> spp. isolates were <italic>B. melitensis</italic> biovar 1 and <italic>B. abortus</italic> biovar 3. The PCR results were the same as the bacteriological method for detecting <italic>Brucella</italic> spp. The sensitivity and specificity of the URS-PCR method make it suitable for detecting <italic>B. abortus</italic> and <italic>B. melitensis</italic>.</p></sec><sec><title>Conclusion</title><p>Quick detection of <italic>B. abortus</italic> and <italic>B. melitensis</italic> can provide the most effective strategies for control of these bacteria. The advantage of this method over other presented methods is that both <italic>B. abortus</italic> and <italic>B. melitensis</italic> are detectable in a single test tube. Furthermore, this method covered 100% of all <italic>B. melitensis</italic> and <italic>B. abortus</italic> biotypes. The development of this URS-PCR method is the first step toward the development of a novel kit for the molecular identification of <italic>B. abortus</italic> and <italic>B. melitensis</italic>.</p></sec>
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