The aim of the study was to assess genotypic and phenotypic diversity among a large number of clinical isolates of Borrelia burgdorferi sensu lato obtained from patients in Slovenia. Plasmid profiles and species identification were determined by PFGE, protein profiles by SDS-PAGE. Of 706 B. burgdorferi sensu lato human isolates 599 (85%) were found to be B. afzelii, 101 (14%) B. garinii and six (1%) B. burgdorferi sensu stricto. The vast majority of strains (605; 86%) were isolated from skin, 58 (8%) from blood, and 43 (6%) from CSF. When analysed by RFLP, B. afzelii strains were unique, while heterogeneity was found within B. garinii and B. burgdorferi sensu stricto species. An unusual plasmid content was found in 52/706 (7%) isolated strains, more often in B. garinii than in B. afzelii strains. A plasmid dimer was found in B. afzelii and B. garinii strains, whereas multiple copies of the large plasmid were associated nearly exclusively with B. garinii strains. Analysis of protein profiles revealed that OspA and OspB are expressed more often by B. afzelii strains, and OspC by B. garinii strains. Heterogeneity of Borrelia strains may play a significant role in the virulence and pathogenesis of the infection. Differences in antigenic components have an important impact on serological testing and vaccine development.