The Lyme Borrelia genospecies Borrelia afzelii and B. garinii have previously been isolated using a culture method in Swedish patients with Lyme borreliosis (LB). There are reports suggesting that the genospecies distribution in human tissue specimens as determined by molecular methods is different from that obtained by culture. In the present study, we developed a nested PCR for detection of Lyme Borrelia-specific DNA in cerebrospinal fluid from Swedish patients with LB. The genospecies were subsequently identified by sequence analysis in a total of 7 PCR-positive patients. Two sequences were identified as B. burgdorferi sensu stricto (s. s.), 1 as B. afzelii and 4 as B. garinii. These are the first reported cases in which B. burgdorferi s. s. has been shown to be the causative agent of human LB in Sweden. The results of our study confirm that the use of direct molecular analytical methods for Borrelia genospecies identification in clinical specimens can provide epidemiological information additional to that obtained by culture.