In the Brazilian Amazon biome, there has been a rise in human spotted fever cases, but still significant knowledge gaps regarding the diversity and epidemiology of the tick–host–Rickettsia relationship. In the herein study, rickettsiae were investigated in ticks from captured live wild hosts in the Amazon biome by PCR targeting a partial sequence of ompB and gltA genes. All 343 ticks were morphologically identified as belonging to five species of the genus Amblyomma. Amblyomma varium (n = 24, 7%) were collected from a Rhinella marina and a Bradypus tridactylus. Amblyomma geayi (n = 51, 15%) were collected from two Bradypus tridactylus. Amblyomma goeldii (n = 116, 34%) were collected from three Tamandua tetradactyla. Amblyomma dissimile (n = 131, 38%) were collected from two Boa constrictor. Amblyomma humerale (n = 21, 6%) were collected from a Chelonoidis spp. Four A. geayi ticks (one female and three males) collected from a three-toed sloth (B. tridactylus) in 2015 were found to be positive for Rickettsia parkeri strain Atlantic Rainforest. The molecular findings herein have confirmed that R. parkeri spotted fever may occur in the Amazon Rainforest associated with A. geayi, expanding the geographical distribution of the R. parkeri strain to the Central Amazon Rainforest.