Abstract： Background Human rickettsioses are worldwide zoonoses and it is not easy to differentiate them from other infectious diseases because of their atypical manifestation. In recent years the number of patients with fever of unknown causes from Hongta District CDC, Yuxi city of Yunnan Province has been increasing significantly in the summer. Diagnosis of scrub typhus was made by local clinicians. In order to ascertain the disease, we undertook a laboratory investigation for such patients from August 18 to 26, 2005.Methods Active surveillance was conducted by Hongta District CDC Yuxi city of Yunnan Province from 2002 to 2004 and basic data were obtained from cases confirmed according to clinical definitions. Average incidences and town-level incidences were calculated during the study periods. Blood samples were analyzed by PCR and serological test. Based on the groEL gene sequences a paired general outer primers (Gro-1 and Gro-2) targeting typhus, spotted fever as well as scrub typhus and two paired inner primers (SF1, SR2 and TF1, TR2) for typhus together with spotted fever and scrub typhus, respectively, were designed to perform a multiplex-nested PCR. Serological assay was carried out by indirect immunofluorescence assay with 7 different rickettsial antigens, i.e., R. mossori, R. sibirica, R. conorii, O. tsutsugamushi,B. quintana, B. henselae and Coxilella burnetii phase Ⅱ Ag.Results Epidemiological surveillance showed that from 2002 to 2004, the average incidences of the scrub typhus or scrub typhus with murine typhus were 222.1/105, 204.3/105 and 109.6/105, respectively. Of 13 blood samples taken during acute stage of illness, 6 showed the amplified products for scrub typhus and the sequenced products showed 100%, 99%, 99%, 99%, 99%, 99% similarity to O. tsutsugamushi Karp but they shared the same deduced amino acid sequences, which indicated 100% identity with the heat shock protein of the O. tsutsugamushi Karp strain. Five yielded PCR products for murine typhus and their corresponding nucleotide sequences exhibited 100%, 100%, 99%, 99% and 99% similarity to R. mossori Wilmington and the analyses of predicted amino acid sequences indicated 100%, 100%,98%, 98% and 98% identity with the heat shock protein of R. mossoriWilmington strain. Of the 8 PCR positive patients, 3 showed a co-infection of scrub typhus with murine typhus. All the 13 serum samples from febrile patients were positive against O. tsutsugamushi and 8 of them were positive against R. mossori. All of the 8 paired specimens had four-fold elevation of antibody against O. tsutsugamushi, and seroconversion for typhus was demonstrated in 3 paired serum samples. Another finding in the study was that a high seropositive prevalence (76.9%) of Q fever was detected.Conclusion It's confirmed that co-prevalence of scrub typhus with murine typhus are occurring in Yuxi city of Yunnan province, China. Other rickettsial diseases also need to be investigated in these areas.