Lyme disease (LD) is a multisystemic inflammatory disease caused by pathogenic spirochetes, belonging to the genospecies complex Borrelia burgdorferi sensu lato (B.b.s.l.). Around the world, distinct species of Ixodes tick vectors transmit different species of Borrelia. Despite the rising recognition and occurrence of tick-borne disease in Latin America, serology has proven to be inconclusive in detecting suspected LD cases. Recently, new B.b.s.l. strains or new related species have been described in Brazil, Uruguay, and Chile. This could explain the lack of confirmatory tests, such as indeterminate Western blots (WBs) and polymerase chain reactions, in detecting suspected LD cases in this region of the world. Future studies will need to determine the extension of novel B.b.s.l. species infections in ticks, reservoirs, and humans in Latin America. The existence of these new Borrelia genomic species should prompt the development of innovative diagnostic and clinical approaches.