AbstractAnaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity ofAnaplasmaspp. andEhrlichiaspp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect therrsanddsbgenes ofAnaplasmaspp. andEhrlichiaspp., respectively. The assays were positive in 27.57% (91/330) of theAnaplasmaspp. and 24.54% (81/330) of theEhrlichiaspp. Of the 91 positiveAnaplasmaspp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S–5S intergenic region. Phylogenetic and distance analyses based on therrsgene allocatedAnaplasmasequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to theA. marginale,A. ovis,andA. capraclades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences ofAnaplasmaspp. detected inNasua nasua,Leopardus pardalis, andCerdocyon thousin Brazil. These sequences were positioned close toA. odocoileisequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinctAnaplasmagenotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected inN. nasuain Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes ofAnaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those inA. marginale,A. ovis,andA. capra.Anaplasmaspp. sequences detected in Xenarthra from São Paulo and were allocated close to those inA. phagocytophilum. The analyses based on thedsbgene grouped theEhrlichiaspp. sequences with sequences ofE. canis(São Paulo, Mato Grosso do Sul, and Pará) andE. minasensis(Rondônia and Pará). The data indicate the occurrence ofE. canisandE. minasensisand two possible newCandidatusspecies ofAnaplasmaspp. in free-living mammals of the Superorder Xenarthra in Brazil.