Background: Malta fever caused by the Brucella spp. is an endemic zoonotic infectious disease in Iran. Evaluation of the disease control is essential for socioeconomic and public health-related concerns. Objectives: This study aimed to evaluate the prevalence of Brucella spp. and the frequency of mutations responsible for resistance to rifampin, streptomycin, and Fluoroquinolones in sheep from Mianeh, Southwest of West Azarbaijan Province, Iran. Methods: A total number of 1, 220 blood samples were collected in Mianeh. Positive samples screened using Rose Bengal Plate Test (RBPT) were subjected to Polymerase Chain Reaction (PCR). Finally, positive samples were investigated for the presence of mutated aminoglycoside adenylyl-transferase-A1 (aadA1) (conventional PCR), DNA gyrase subunit A (gyrA), topoisomerase IV subunit C (parC) (MAMA PCR for both) and RNA polymerase -subunit (rpoB) (MAS PCR) responsible for antibiotic resistance. Results: There were 41 Brucella abortus Cell Surface Protein 31 Positive (BCSP31+) samples out of 111 RBPT positive samples which 15% (n = 7) of them were obtained from vaccinated sheep. Of the total BCSP31+ samples 9 were carrying mutated aadA1, 3 carrying mutated parC and only one sample carrying mutated rpoB. None of the samples were positive for mutated gyrA. Conclusions: The prevalence of Brucellosis in livestock in the city of Mianeh has been higher than in other areas in Iran, and some antibiotic resistance genes have been identified in livestock specimens. It can be concluded that due to the relatively good efficacy of the vaccines, the need for vaccination in all livestock farms of this city is important.