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Brucellosis is one of the most important and widespread bacterial zoonoses worldwide. Cases are reported annually across the range of known infectious species of the genus <i>Brucella.</i> Globally, <i>Brucella melitensis</i>, primarily hosted by domestic sheep and goats, affects large proportions of livestock herds, and frequently spills over into humans. While some species, such as <i>Brucella abortus</i>, are well controlled in livestock in areas of North America, the Greater Yellowstone Ecosystem supports the species in native wild ungulates with occasional spillover to livestock. Elsewhere in North America, other <i>Brucella</i> species still infect domestic dogs and feral swine, with some associated human cases. <i>Brucella</i> spp. patterns vary across space globally with <i>B. abortus</i> and <i>B. melitensis</i> the most important for livestock control. A myriad of other species within the genus infect a wide range of marine mammals, wildlife, rodents, and even frogs. Infection in humans from these others varies with geography and bacterial species. Control in humans is primarily achieved through livestock vaccination and culling and requires accurate and rapid species confirmation; vaccination is <i>Brucella</i> spp.-specific and typically targets single livestock species for distribution. Traditional bacteriology methods are slow (some media can take up to 21 days for bacterial growth) and often lack the specificity of molecular techniques. Here, we summarize the molecular techniques for confirming and identifying specific <i>Brucella</i> species and provide recommendations for selecting the appropriate methods based on need, sensitivity, and laboratory capabilities/technology. As vaccination/culling approaches are costly and logistically challenging, proper diagnostics and species identification are critical tools for targeting surveillance and control.