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Diagnosis of human Lyme borreliosis is usually based on serology, which has a number of pitfalls. In the early phase of the disease serology can still be negative, whereas false-positive results are also common. The interpretation of confirmatory Western blot tests is not always easy. Furthermore, routine serology cannot discriminate between active and past infection. In addition, recombinant antigens are being introduced to improve serologic tests. New developments in the diagnosis of Lyme disease are the development of PCR tests. This review gives an overview of the molecular diagnostic possibilities of Lyme borreliosis, mainly by PCR, and describes some interesting possibilities for future serology.