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Brucellosis is an important zoonosis and a significant cause of reproductive losses in animals. Abortion, placentitis, epididymitis, and orchitis are the most common clinical manifestations in animals. In humans, brucellosis is a debilitating and chronic disease, which may affect a variety of organs. Clinical diagnosis of brucellosis is not easily achieved. Laboratory testing is therefore very important for a correct identification of the disease in humans and for the detection and confirmation in animals. Definitive diagnosis is normally done by isolation and identification of the causative agent. While definitive, isolation is time-consuming, must be performed by highly skilled personnel, and it is hazardous. For these reasons, serological tests are normally preferred. Brucellosis serology have advanced considerably in the last decades with very sensitive and specific new tests available. Modern genetic characterization of Brucellae using molecular DNA technology have been developed. Several PCR-based assays have been proposed, from the rapid recognition of genus to differential identification of species and strains. This review describes bacteriological, serological, and molecular methods used for the diagnosis of human and animal brucellosis.