As a part of a larger survey a total of 599 Ixodes ricinus ticks collected from four locations (Neustadt an der Waldnaab, Amberg, Poppenricht and Lintach) in the north of the Upper Palatinate in Bavaria were investigated for infection with Borrelia burgdorferi sensu lato (s. l.) species using the 5S-23S intergenic spacer of rRNA gene as a target in a nested PCR (Rijpkema et al., 1995) and a sequencing method. Overall, 15.8% ticks were infected with B. burgdorferi s. I. Borrelia afzelii (43.1%) was the predominant genospecies, followed by Borrelia valaisiana (14.7%), Borrelia garinii (13.7%) and Borrelia burgdorferi sensu stricto (6.3%). Also Borrelia spielmanii was found (1.1%). Of the infected ticks, 21.1% harbored multiple infections with B. burgdorferi s. I. The highest number of infected ticks was found in Amberg (22.5%) and the lowest number in Neustadt an der Waldnaab (11.9%). In Poppenricht and Lintach, the numbers of infected ticks were 12% and 18.7%, respectively. Human pathogenic Borrelia species were found to be prevalent at each study site thus representing the potential risk for people living and visiting these areas.