The primary objective of this study was to determine whether patients with putative late-onset Lyme arthritis based upon synovial fluid Borrelia burgdorferi IgM and IgG immunoblot testing offered by commercial laboratories satisfied conventional criteria for the diagnosis of Lyme arthritis. Secondary objectives included assessing the prior duration and responsiveness of associated antibiotic therapy. We conducted a retrospective analysis of 11 patients referred to an academic medical center infectious disease clinic during the years 2007 to 2009 with a diagnosis of Lyme disease based upon previously obtained synovial fluid B. burgdorferi immunoblot testing. Ten of the 11 (91%) patients with a diagnosis of late-onset Lyme arthritis based upon interpretation of synovial fluid B. burgdorferi immunoblot testing were seronegative and did not satisfy published criteria for the diagnosis of late-onset Lyme arthritis. None of the 10 patients had a clinical response to previously received antibiotics despite an average course of 72 days. Diagnosis of Lyme arthritis should not be based on synovial fluid B. burgdorferi immunoblot testing. This unvalidated test does not appear useful for the diagnosis of Lyme disease, and this study reinforces the longstanding recommendation to use B. burgdorferi immunoblot testing only on serum samples and not other body fluids. Erroneous interpretations of "positive" synovial fluid immunoblots may lead to inappropriate antibiotic courses and delays in diagnosis of other joint diseases.