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Aim of this study is to report a laboratory investigation performed following the isolation of <i>Brucella ovis</i>, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for <i>B. ovis</i> BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while <i>B. ovis</i> was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of <i>B. ovis</i> infection is still unknown, particularly in our country where the disease is considered endemic.