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The 4 Borrelia burgdorferi sensu lato isolates obtained from 1. Ricinus ticks collected in the natural foci in Russia and Ukraine, having an unusual RFLP Msel-pattern, were studied using sequencing rrfA-rrlB spacer and rrs gene. The Ir-5215 isolate from the tick collected in southern Ukraine represented recently described genospecies B. spielmanii pathogenic for humans. The three atypical isolates Ir-3519, Ir-4721, and Ir-4812 had 100% identity with the sequence of the atypical European B. burgdorferi sensu stricto strains. They constituted a subgroup of the B. burgdorferi sensu stricto on the grounds of Multilocus sequence analysis (MLSA). These data can be indicative of the genetic heterogeneity of the current group B. burgdorferi sensu stricto.