Strain 25015 is an atypical tick isolate that belongs to a distinct genomic group (DN127) within the general taxon Borrelia burgdorferi sensu lato. Similarities between this strain and a white-footed mouse isolate from Illinois, strain CT39, have been reported. In the course of isolating B. burgdorferi sensu lato in culture from Slovenian patients, 9 isolates were identified with the same genetic profiles as strains 25015 and CT39, as evidenced by restriction enzyme MluI digestion patterns of genomic DNA. The aim of the present study was to molecularly characterize all 11 isolates to examine the extent of their genotypic and phenotypic similarity. The results of molecular studies suggest a close relationship between the patient isolates and strains 25015 and CT39. However, CT39 and several patient isolates possessed unique characteristics that reflect their discrete ontogeny.