| Summary: | <i>Borrelia burgdorferi</i> sensu lato (Bbsl) spirochetes thrive in sylvatic transmission cycles infecting vertebrates and their ticks. Rodents and ticks of the genus <i>Ixodes</i> are important hosts of these spirochetes globally. Although evidence suggests that <i>Borrelia burgdorferi</i> sensu stricto does not exist in South America, genospecies of the group (Bbsl) can be found in this region but have been poorly characterized from a genetic viewpoint, and data on their ecoepidemiology are still incipient. Aiming to detect the natural foci of <i>Borrelia</i> in Brazil, we targeted small mammals inhabiting seven forests fragments during a period of three years (2015–2018). Organs (lung) from two <i>Oligoryzomys</i> rodents over a total of 382 sampled mammals were positive, and we performed a molecular characterization of 10 borrelial genes to achieve a robust analysis. Phylogenetic trees inferred from 16S rRNA, <i>flaB</i>, <i>ospC</i>, and seven MLST loci (<i>clpA</i>, <i>nifS</i>, <i>pepX</i>, <i>pyrG</i>, <i>recG</i>, <i>rlpB</i>, and <i>uvrA</i>) support the characterization of a novel genospecies of Bbsl that we herein name “<i>Candidatus</i> Borrelia paulista” Rp42. Remarkably, “<i>Ca</i>. B. paulista” is phylogenetically related to <i>Borrelia carolinensis</i>, a genospecies that infects <i>Ixodes</i> ticks and cricetid rodents in North America. A previous study performed in the same area identified <i>Ixodes schulzei</i> feeding on <i>Oligoryzomys</i> rodents. Although this tick species could be considered a probable host for this novel <i>Borrelia</i> sp., further research is needed to confirm this hypothesis.
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