Direct detection of Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, is the most reliable laboratory diagnostic tool. Several methods have been developed for direct detection of B. burgdorferi in infected vectors, host tissues, and clinical specimens from patients with Lyme borreliosis. These include microscope-based assays, antigen detection assays, in vitro cultivation, and nucleic acid-based detection of B. burgdorferi. The sensitivity and specificity of these methods depend on various factors and are also variable among laboratories. To date, only in vitro cultivation of B. burgdorferi has been widely accepted to confirm clinical diagnosis of Lyme borreliosis. Nevertheless, various polymerase chain reaction-based molecular assays have shown increasing significance in the laboratory diagnosis of Lyme borreliosis because of their high sensitivity, specificity, and capability for quantification and typing of spirochetes in clinical specimens. In this review, the currently available methods for direct detection of B. burgdorferi in clinical samples and quantitative analysis of spirochete load in different biological sources are discussed.