The Borrelia culture collection at the Vector Laboratory of the Gamaleya Research Institute of Epidemiology and Microbiology, Moscow dates from 1983. Today, this collection consists of almost 1,200 primary isolates from different sources from various geographic regions. A PCR–RFLP analysis of isolates from Russia and neighboring countries has shown that they include primarily genospecies of B. garinii and B. afzelii, which are widespread and epidemiologically significant, along with B. burgdorferi sensu stricto, B. valaisiana, B. lusitaniae, and B. spielmanii (group A14S). Considerable genetic heterogeneity of B. afzelii has been revealed by comparing the sequences of the rrf (5S)-rrl (23S) ribosomal intergenic spacer in 139 primary isolates obtained from ixodid ticks and from several species of small mammals. All these isolates were previously identified as B. afzelii by PCR–RFLP analysis of amplicons of the 5S–23S rRNA spacer region. Two genomic subgroups have now been identified. The majority of the isolates have a high degree of homology to the most widespread genomic subgroup of B. afzelii, strain VS461 (98.8–99.6%). The remainder have a high degree of homology to the other genomic subgroup B. afzelii, strain NT28 (98.4–99.6%). Several genovariants have been identified within each subgroup with the degree of nucleotide homology within most of them reaching 100%. Seven of ten genovariants belong to subgroup VS461, three genovariants belong to subgroup NT28. Seven allelovariants have been detected by comparing the sequences of a p66 gene fragment (246–377 bp) in 45 B. afzelii isolates. Three of seven B. afzelii VS461 genovariants are apparently common in Eurasian natural foci among different species of vectors and reservoir hosts, with one genovariant probably circulating mainly in foci where I. persulcatus is the main vector of Borrelia infection. The remaining four genovariants of this subgroup appear to be associated with I. ricinus ticks and circulate mainly in Europe. The distribution of the three genovariants belonging to B. afzelii NT28 subgroup needs further study. Nucleotide sequences of approximately 100 isolates of different Borrelia genospecies have been deposited in GenBank.