Diversity of Lyme disease spirochetes and their protein encoding genes play a major role in pathogenesis and diagnosis of Lyme borreliosis. In a survey of 72 CSF isolates from Germany, we found 15 (20.5%) Borrelia afzelii, 39 (53.4%) B. garinii, and 18 (24.7%) B. burgdorferi sensu stricto ; among 160 skin isolates, B. afzelii was present in 107 (66.8%), B. garinii in 39 (24.3%), B. burgdorferi sensu stricto in 10 (6.3%), and the new species B. spielmanii in 4 (2.5%). A small number of isolates from synovial fluids (n=6) revealed heterogeneity of the causative strains (2 B. burgdorferi sensu stricto , 2 B. afzelii, and 2 B. garinii). Among 507 PCR-positive ticks from Southern Germany, 22% harboured B. burgdorferi sensu stricto , 25% B. afzelii, 34% B. garinii, 16% B. valaisiana, and 6% B. spielmanii. Sequence identities among major immunodominant proteins (DbpA, VlsE, OspC, OspA, BmpA, p83/100, p58, and flagellin) from the three main human pathogenic species increase from 40–44% to 96–97%. Comparison of dbpA sequences with ospC sequences from a panel of 59 strains revealed all kinds of cross-connections indicating processes of lateral gene transfer. The extent of sequence identities among the dbpA genes decreased from the DNA (67%) to the AA level (44%) about 23%, and ospC sequence identities differed about 10%, an indication that both proteins, but especially DbpA, play a role in immune escape. A combination of four DbpAs (including two different B. garinii DbpAs) was crucial for sensitive serodiagnosis. The use of primarily in vivo-expressed recombinant proteins like OspC, DbpA, and VlsE significantly improved antibody detection. VlsE and DbpA are the most sensitive antigens for IgG antibody detection. OspC is not only the immunodominant protein of the early (IgM) response in the infected host, but it is also crucial for host infectivity. It is controversial to assume that OspC is necessary for borrelial invasion of the tick salivary glands. We found that an OspC-positive B. afzelii wild type strain disseminated from the midgut to the salivary glands, while an OspC-negative mutant was only present in the tick midguts. Colonization of salivary glands by the mutant was restored by complementation of this strain with a plasmid construct that constitutively expresses the wild type ospC. Thus Borrelia strains might also differ in their potential to disseminate from the gut to the ticks' salivary glands.