Thirty years after it was introduced, whole DNA/DNA hybridization (WDDH) is still considered as the gold standard for bacterial taxonomy. However, WDDH has serious limitations, as it requires large volumes of cultures, is a highly fastidious and time consuming procedure, lacks of reproducibility, and requires pairwise comparisons between unknown genome and genomes from previously described species. These limits have hampered progress in Borrelia taxonomy. With the development of sequencing, it is inviting to propose MLSA as an alternative to WDDH. We chose fragments from seven loci: rrs (16S rRNA), flaB, groEL, hbb, recA, ospA, and the internal transcribed spacer rrf-rrl (ITS). We sequenced these loci for 2–4 representative Borrelia burgdorferi sensu lato strains from each known species present on both the European and North American continents. Phylogenetic trees were built from sequences of each locus, confirming that lateral transfer was a rare event, and from the concatenated sequence of the seven loci from each strain. Genetic distances were calculated from concatenated sequences and compared to those obtained by WDDH (both published and unpublished). The two methods were strictly correlated. A precise cutoff of MLSA-based genetic distances (0.021) allowed us to delineate new species more safely and accurately than the usual 70% DNA relatedness cutoff. We have confirmed in this way a recently described sixth species in Europe, B. spielmanii, whose reservoirs are dormice. Three new nonpathogenic Borrelia species are delineated in California, genomospecies 1, 2, and 3. Finally, the phylogenetic tree obtained from the concatenated sequences from 42 isolates (from both Europe and North America) shows 12 clusters, each corresponding to a species. These clusters form two main groups. One group comprises only European species, whereas the other group comprises B. burgdorferi sensu stricto isolates from Europe and the USA, including both Borrelia spp. “border line” to B. burgdorferi sensu stricto. This clustering confirms our previous hypothesis of the American origin of B. burgdorferi sensu stricto and closely related isolates that have been imported recently into Europe from the USA.