Abstract: Objective To investigate the infection status and epidemiological features of spotted fever group Rickettsiae (SFGR) in patients with fever of unknown origin(FUO) in Hainan Province, and analyze the characteristics of detection of rickettsial disease,so as to establish a rapid strong specificity, high sensitivity and good repeatability laboratory testing system which would provide scientific basis for prevention and control of the spotted fever. Methods Nested polymerase chain reaction(Nested PCR,) multiple real-time fluorescence quantitative PCR, indirect immunofluorescence and pathogen isolation culture methods were conducted respectively to inspect the blood samples of patients with FUO. Results Nested PCR was performed to detect the specific fragment of groEL gene in 399 blood specimens of fever patients.The results showed that 17 samples contained spotted fever group and typhus group rickettsiae, 61 samples contained orientia tsutsugamushi. Meanwhile, the result of multiplex real-time fluorescence quantitative PCR test, showed that 2 samples contained spotted fever group rickettsiae, 6 samples contained typhus fever group rickettsiae, 49 samples contained orientia tsutsugamushi. Furthermore,when indirect immunofluorescence assay were performed, SFGR IgM were observed in 19 samples and IgG were observed in 148 samples. Typhus fever group rickettsiae IgM were observed in 29 samples, and IgG in 169 samples. However, no cytopathic effect and negative results were showed in the test isolated and cultured the blood specimens with L929 monolayer cells. Conclusions Four kinds of laboratory testing methods could further clarify the epidemic characteristics of spotted fever from aspects of etiology, serology and molecular biology,provide plenty data for diagnosis and protection.