Abstract: Objective To investigate the natural infection of Orientia tsutsugamushi in patients with fever of unknown origin in Yunnan,China.Methods Blood samples were collected from the patients with fever of unknown origin for the detection of antibody (Ab) of Ot with IFA and amplification and sequencing of partial sta56 segment of Ot with nested-polymerase chain reaction (nPCR),the homology with other known sequences were analyzed.Results In 79 patients with fever of unknown origin,the sero-positive rate was 48.10% for IgG antibody and 3.80% for sta56 gene of Ot.Sequence analysis showed that sequence 12-17 shared higher nucleotide homology (99.8%) with O3 strains from Japan,sequence 12-9 shared high nucleotide homology with some Taiwan Ot strains,such as KM05 and TW45R,all reaching up to 99.8% and sequence 11-6 shared high nucleotide homology with some Taiwan Ot strains,such as KM05 and TW45R,all reaching up to 99.8%.Phylogenetic analysis showed that sequences 12-17,12-9 and 11-6 all belonged to Karp genotype of Ot.Conclusion There was Karp genotype Ots causing scrub typhus in Yunnan,and their origins might be complex in genetic evolution.