Spotted fever (SF) is a tick-borne disease associated with Rickettsia spp.. In the Pampa biome, Southern Brazil, cases of SF seem to be strongly linked with the practice of hunting wild animals. An investigation of rickettsiae in tick species found on wild animals could provide more information regarding the rickettsiosis enzootic cycle. The aim of this study is to describe the results of a molecular survey of Rickettsia spp. in the Neotropical deer tick, Haemaphysalis juxtakochi Cooley, 1946 (Acari: Ixodidae), from the Brazilian Pampa. Ticks were obtained from 14 road-killed gray brocket deer, Mazama gouazoubira (Artiodactyla: Cervidae), found in nine different municipalities of Rio Grande do Sul state, Southern Brazil. Ticks were processed individually to obtain genomic DNA, and then Rickettsia spp. was investigated using a set of PCR reactions that amplified the rickettsial fragments of the gltA, ompA, and htrA genes. Of the 24 tick samples tested, DNA of Rickettsia parkeri sensu stricto (s.s.) was found in 11 H. juxtakochi specimens collected in two different areas of the Brazilian Pampa. This is the first report of R. parkeri s.s. (the main agent associated with SF in the Uruguayan, Argentinian, and Brazilian Pampa) in H. juxtakochi ticks. These findings indicate that R. parkeri s.s. may be much more common and widely distributed in the Pampa biome than previously assumed. Moreover, H. juxtakochi ticks and gray brocket deer could participate in the potential spillover of R. parkeri s.s. from endemic to non-endemic areas in the South American Pampa.