Ticks (especially those in the family Ixodidae) are the primary vectors of the infectious agent Borrelia burgdorferi sensu lato, which causes Lyme disease. To determine the potential role of mites as vectors of B. burgdorferi sensu lato, mites were collected from wild rodents in the BaŽantula region of North Moravia (Czech Republic). These samples were examined for the presence of Borreliae by using DFM and PCR methods. Mites positive for the presence of DNA of B. burgdorferi sensu lato were determined as members of the families Haemogamasidae and Parasitidae. One sample from a mite of genus Haemogamasus was successfully isolated, and the specimen was confirmed as B. afzelii by using PCR-RFLP and by gradient SDS-PAGE. This suggests the possible participation of gamasid mites in borrelial circulation in nature and also points to the utility of further such studies to identify potential vectors (other than ticks) of the spirochete. Vector-Borne Zoonotic Dis. 5, 227–232.