Coxiella burnetii is a globally distributed zoonotic γ-proteobacterium with an obligatory intracellular lifestyle. It is the causative agent of Q fever in humans and of coxiellosis among ruminants, although the agent is also detected in ticks, birds, and various other mammalian species. Requirements for intracellular multiplication together with the necessity for biosafety level 3 facilities restrict the cultivation of C. burnetii to specialized laboratories. Development of a novel medium formulation enabling axenic growth of C. burnetii has facilitated fundamental genetic studies. This review provides critical insights into direct diagnostic methods currently available for C. burnetii. It encompasses molecular detection methods, isolation, and propagation of the bacteria and its genetic characterization. Differentiation of C. burnetii from Coxiella-like organisms is an essential diagnostic prerequisite, particularly when handling and analyzing ticks.