This study reports the evaluation of the technical and clinical validation of the O-DiaBorburg kit (DIA), Borrelia burgdorferi PCR kit, ISEX (GENE), and Borrelia burgdorferi sensu lato Real-TM (SAC) for the diagnosis of neuroborreliosis in cerebrospinal fluid based on both Borrelia DNA and CSF samples from patients with clinical suspicion of neuroborreliosis. This validation study was done by analysing the kits on the Rotorgene Q (RGQ), CFX96, and LightCycler480 (LC480). For all kits, the linear range was larger on RGQ than on CFX96 and LC480. A good reproducibility was obtained for all assays on all instruments. Storage at -20 °C resulted in a decreased reproducibility for SAC. Results of the limit of detection (LOD95) experiments indicated a better sensitivity than described in the kit insert for all kits on all PCR platforms. No cross-reactivity was found for genetically related organisms nor for other pathogens which may be present in CSF. All species of the Borrelia burgdorferi sensu lato complex were detected with the GENE and SAC kits. The DIA kit failed to detect B. lusitaniae. The results seemed to indicate a better overall performance for the GENE kit on RGQ. However, its diagnostic value could not be confirmed in the clinical validation study, wherein none of the 103 CSF samples from clinical neuroborreliosis cases showed a positive real-time PCR result with the GENE kit analysed on RGQ.