California has a remarkable diversity of squirrel and chipmunk species (sciurids), and five named and several unnamed genospecies in the Borrelia burgdorferi sensu lato group (BBSL) of bacteria as well, many of which utilize sciurids as reservoirs. We investigated the prevalence, spatial distribution, and diversity of BBSL in sciurids of California by literature search, PCR of 585 ear tissue samples from 15 sciurid species prospectively collected across 19 California counties, and DNA sequencing when possible. Seven publications documented BBSL infections in western gray squirrels (Sciurus griseus), fox squirrels (Sciurus niger), eastern gray squirrels (Sciurus carolinensis), Douglas squirrels (Tamiasciurus douglasii), and redwood chipmunks (Tamias ochrogenys) in northern California. Prospective sampling added new BBSL infection records for long-eared chipmunks (Tamias quadrimaculatus), Allen's chipmunks (Tamias senex), and Siskiyou chipmunks (Tamias siskiyou). Infection was detected in the Mendocino, North Coast, West Sierra, and Central Valley regions of California. The overall PCR prevalence was 9.4% (n = 585), and exceeded 40% (n = 84) in Mendocino and farther north along the Pacific coast. Redwood (40.7%, n = 81) and Siskiyou (22.2%, n = 18) chipmunks had the highest prevalence of BBSL infection. BBSL infections were associated with arboreal and semiarboreal sciurid species and species occurring in conifer forests. Western gray squirrels and Allen's chipmunks in Humboldt County and redwood chipmunks in Mendocino County were infected with B. burgdorferi sensu stricto, while we identified Borrelia bissettiae in Douglas squirrels and Siskiyou chipmunks in Humboldt and Del Norte Counties. This indicates that further study of sciurids can aid in describing the ecology of BBSL in California.