Tick-borne relapsing fever (TBRF) is endemic in mountainous regions of the western United States. In California, the principal agent is the spirochete Borrelia hermsii, which is transmitted by the argasid tick Ornithodoros hermsi. Humans are at risk of TBRF when infected ticks leave an abandoned rodent nest in quest of a blood meal. Rodents are the primary vertebrate hosts for B. hermsii. Sciurid rodents were collected from 23 sites in California between August, 2006, and September, 2008, and tested for serum antibodies to B. hermsii by immunoblot using a whole-cell sonicate and a specific antigen, glycerophosphodiester phosphodiesterase (GlpQ). Antibodies were detected in 20% of rodents; seroprevalence was highest (36%) in chipmunks (Tamias spp). Seroprevalence in chipmunks was highest in the Sierra Nevada (41%) and Mono (43%) ecoregions and between 1900 and 2300 meters elevation (43%). The serological studies described here are effective in implicating the primary vertebrate hosts involved in the maintenance of the ticks and spirochetes in regions endemic for TBRF.