The diversity of Borrelia burgdorferi subspecies (sensu lato) in Europe does not allow to simply adopt the US definitions and tests which were established for B. burgdorferi sensu stricto. Routine detection of Borrelia-specific antibodies now includes specific antigens of the subspecies B. afzelii and B. garinii. Thus, the 2-step serology of screening by ELISA and confirmation by immunoblot remains the most important diagnostic test of lyme borreliosis. The detection of borrelia by PCR or culture is of lesser importance due to the limited sensitivity, despite of the high specificity. In clinical practice, the risk of exposure to ticks and the stage of the clinical presentation guide the choice of appropriate diagnostic procedures and their interpretation.