This study reports the development and evaluation of an internally controlled real-time PCR targeting the ospA gene for detection of Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and Borrelia valaisiana. DNA was extracted using QIAamp DNA Blood Mini kit columns. DNA from 33 B. burgdorferi sensu lato strains reacted in the assay, whereas no reactivity was observed with DNA from four relapsing fever Borrelia spp., 11 unrelated spirochaetes, and 31 unrelated microorganisms. The quantitative sensitivity of the assay was 1-10 fg of Borrelia DNA and one to five cultured Borrelia spirochaetes. Cerebrospinal fluid (CSF) specimens from 70 patients sent for routine testing for neuroborreliosis, and three CSF specimens containing B. garinii were also tested. Positive PCR results were obtained with all three culture-confirmed neuroborreliosis specimens, five of ten neuroborreliosis specimens with specific antibodies in CSF and pleocytosis, none of nine specimens from possible cases of early neuroborreliosis (antibodies in serum, CSF pleocytosis, no antibodies in CSF), one of 15 specimens from patients with active or past Lyme disease with neurological signs (antibodies in serum, no pleocytosis or antibodies in CSF), and none of 36 specimens from patients without Lyme borreliosis (no antibodies in serum or CSF). Overall, the real-time PCR assay enabled sensitive and specific detection of all B. burgdorferi sensu lato species tested. The PCR had a sensitivity of 50% in patients with neuroborreliosis. The main diagnostic role of the assay could be to confirm neuroborreliosis in patients for whom the diagnosis is doubtful.