Background: Scrub typhus is a leading cause of febrile illness in Laos and accounts for a high burden of disease. There have been no previous studies on the causative agent, Orientia tsutsugamushi, in vector mites ("chiggers") or their small mammal hosts in Laos. Materials and Methods: Small mammals and free-living chiggers were trapped in districts of Vientiane Province and Capital. Tissues were tested for O. tsutsugamushi by PCR and serum for IgG to O. tsutsugamushi by immunofluorescence assays (IFAs). Chiggers removed from small mammals and collected in their free-living stage using black plates were identified and tested for O. tsutsugamushi by PCR. Results: Over an 18-month period, 131 small mammals of 14 species were collected in 5 districts. Seventy-eight of 131 small mammals were infested with chiggers, but all tissues were O. tsutsugamushi PCR negative. Eighteen species of chigger were identified and 1,609 were tested by PCR. A single pool of chiggers tested O. tsutsugamushi positive. Sera from 52 small mammals were tested by IFA, with 16 testing positive. Conclusions: These are the first molecular and serological data on O. tsutsugamushi in chiggers and small mammals in Laos. Further studies are needed to better understand the key vector species and ecology of scrub typhus in areas with high disease incidence in Laos.