Orientia tsutsugamushi, the causative agent of scrub typhus, is widely prevalent in the Asia–Pacific region and causes an estimated 1 million cases per year (1). It is characterized by dramatic genetic diversity (2). In the 1960s, complement fixation initially identified O. tsutsugamushi as Karp, Gilliam, and Kato types (3), which are widely used as tested antigens in serologic assays. Since then, >20 antigenic variants of O. tsutsugamushi have been identified by immunologic and molecular methods (2). The 56-kDa type-specific antigen (TSA), which is one of the major immunogens of the agent and is associated with pathogenesis, has been commonly used for type designation (4–6). Scrub typhus is a traditional tropical rickettsiosis. However, since 1986, it has emerged and spread rapidly in temperate zones; the epidemic season is mainly in autumn and winter (7,8). In the late 1990s, the district of Tai’an, west of Shandong Province, northern China (116°20′–117°59′E, 35°38′–36°28′N), became a new epidemic area of autumn–winter type scrub typhus (9); however, few epidemiologic and molecular investigations of the transmission cycle of the disease have been conducted in this area. We therefore investigated the prevalence of O. tsutsugamushi infection among rural domestic rodents in the newly developed epidemic area, evaluated the genotypic diversity according to the variations in the 56-kDa TSA gene, and explored the genetic relationship between strains circulating among domestic rodents and those infecting humans.