Lyme disease is a common tick-transmitted infection caused by spirochetes in the Borrelia burgdorferi sensu lato complex. Borrelia burgdorferi is the primary cause of Lyme disease in the United States, while B. burgdorferi, B. garinii and B. afzelii cause Lyme disease in Europe. Although Lyme disease is primarily diagnosed using clinical and serologic criteria, molecular testing may play an important adjunctive role in detection and have contributed greatly to our knowledge of Lyme disease epidemiology. Molecular tests also increase our ability to detect the presence of co-infecting organisms and allow for detection of previously unknown organisms, such as Borrelia mayonii. This presentation will provide an overview of the available molecular methods for diagnosing Lyme disease and discuss future applications of these technologies.