Rickettsiosis is a potentially fatal tick borne disease. It is caused by the obligate intracellular bacteria Rickettsia, which is transferred to humans through salivary excretions of ticks during the biting process. Globally, the incidence of tick-borne diseases is increasing; as such, there is a need for a greater understanding of tick–host interactions to create more informed risk management strategies. Flinders Island spotted fever rickettsioses has been identified throughout Australia (Tasmania, South Australia, Queensland and Torres Strait Islands) with possible identifications in Thailand, Sri Lanka and Italy. Flinders Island spotted fever is thought to be spread through tick bites and the reptile tick Bothriocroton hydrosauri has been implicated as a vector in this transmission. This study used qPCR to assay Bothriocroton hydrosauri ticks collected from Tiliqua rugosa (sleepy lizard) hosts on mainland South Australia near where spotted fever cases have been identified. We report that, although we discovered Rickettsia in all tick samples, it was not Rickettsia honei. This study is the first to use PCR to positively identify Rickettsia from South Australian Bothriocroton hydrosauri ticks collected from Tiliqua rugosa (sleepy lizard) hosts. These findings suggest that B. hydrosauri may be a vector of multiple Rickettsia spp. Also as all 41 tested B. hydrosauri ticks were positive for Rickettsia this indicates an extremely high prevalence within the studied area in South Australia.