Information about spotted fever group (SFG) rickettsiae in southern China remains sparse. A specific and sensitive real-time PCR assay for detection of SFG rickettsiae was established and used to detect the prevalence rate of SFG rickettsiae in Yunnan Province, China. The limit of detection (LOD) of our real-time PCR was 200 copies per reaction, which is more sensitive than the previously developed nested PCR assays for Rickettsia. We tested 265 blood samples (127 goats, 78 dogs, and 60 cattle) collected from Yunnan Province using the real-time PCR assay and revealed that the prevalence of SFG rickettsiae among dogs, cattle, and goats were 14.10%, 23.33%, and 24.41%, respectively. The SFG rickettsiae detected in animals in Yunnan Province were classified into two genotypes: a unique group that is different from all known SFG rickettsiae species, and R. heilongjiangensis.