Diseases caused by Rickettsiales bacteria are a global public health problem. To better understand the diversity and origins of Rickettsiales infection in humans and animals, we sampled 134 febrile patients, 173 rodents and 43 shrews, as well as 358 ticks, from two cities in Jiangsu and Jiangxi provinces, China. Our data revealed a relatively high prevalence of scrub typhus cases in both localities. In addition, both serological tests and genetic analysis identified three patients infected with Anaplasma bovis, Rickettsia monacensis, and Orientia tsutsugamushi bacteria. Molecular epidemiological investigation revealed the co-circulation of multiple species of Rickettsiales bacteria in small mammals and ticks in both provinces, potentially including novel bacterial species. In sum, these data demonstrate the ongoing importance of Rickettsiales infection in China and highlight the need for the regular surveillance of local arthropods, mammals and humans.