Introduction and Objectives: The inoculum found in patients with brucellosis is normally very low. Due to the sharp decline in PCR inhibitors in serum from blood, this sample is preferred for the diagnosis of brucellosis. This study aimed to assess various primers for using in PCR on animals and human serum samples. Materials and Methods: In total, 38 human serum samples and 30 sheep serum samples in the acute phase of brucellosis were collected. Primers B4-B5, F4-R2 and JPF-JPR are able to detect all species of Brucella. After Software surveys of primer B4-B5, minor changes was done on its sequence. DNA from all samples was separated by boiling and using the primer pairs and PCR was performed on all samples. Also, with each PCR, a positive and a negative control were set up. Results: From 68 samples which were tested by PCR with B4-B5, F4-R2 and JPF-JPR primers, 54 cases (79/41%), 44 cases (64/70%) and 35 cases (51/47%) of them were positive, respectively. From human samples, 33 (86/84%), 27 (71/05%) and 22 cases (57/89 %) with each primer were positive, respectively. Also, from animal samples 21 (70%), 17 (56/67 %) and 13 cases (43/33%) with each primer were positive, respectively. Conclusion: B4-B5 primer in the diagnosis of brucellosis in serum samples when boiling method for DNA extraction was used has highest sensitivity. Lower sensitivity of F4-R2 and JPF-JPR primers is probably due to the presence of inhibitors in serum and low levels of bacterial in serum

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