Rickettsia parkeri rickettsiosis is a vector-borne zoonosis that occurs in some countries of the American continent. Following the first description and determination of the pathogenicity to humans in 2004 in USA, this bacterium has been reported in several South American countries. Human cases have been diagnosed in both Uruguay and Argentina in the past years. This study consisted in a serosurvey of 1000 domestic dogs living in the endemic area of rickettsiosis in Uruguay, where Amblyomma triste is the tick vector. Sera were analyzed by Indirect Immunofluorescence Assay (IFA), against antigens of three different rickettsial species: R. rhipicephali, R. felis and R. parkeri. It was determined that 20.3% of the dogs had antibodies that reacted to at least one of the three species tested, taking as cut off ≥64 titers. Furthermore, 140 of the seropositive dogs (14%) had a titer at least 4 times higher to R. parkeri than those of any of the other species, thus, it was considered that the immune response was stimulated by that species in particular. This is the first serological survey in primary hosts for adults of A. triste in Uruguay, and therefore the first prevalence values are reported. Adult A. triste ticks collected from the environment as well as from dogs were analyzed by PCR in order to confirm the current circulation of the agent in the area. In this matter, two out of 28 ticks from dogs, and 3 out of 53 ticks from the environment were positive, and the corresponding sequence analysis revealed 100% similarity with R. parkeri strain maculatum.