The European red squirrel (Sciurus vulgaris) has long been suspected to be a reservoir host of the agents of Lyme borreliosis, in particular B. burgdorferi sensu stricto (s.s.). However, very few data support this hypothesis. Hereafter, we investigated the infections with B. burgdorferi genospecies in road-killed red squirrels collected across France. We also characterized the diversity of hard tick species collected from a subsample of hosts. DNA of B. burgdorferi genospecies were detected and identified from PCR products in ear biopsies using reverse line blot hybridization. Variation in prevalence was investigated according to biogeographic areas (Mediterranean, Atlantic, Continental, and Alpine), season, sex, relative age, and body mass from 273 squirrels collected 2003–2010. Among the 746 identified tick specimens, no adult was observed, 63% were nymphs, and 37% were larvae all belonging to the species Ixodes ricinus except one nymph identified as I. trianguliceps. Overall, no squirrels of Mediterranean origin and no unweaned juveniles were found infested by hard ticks. Only season explained variation in I. ricinus abundance on squirrels, with more ticks present in spring to summer than in autumn to winter. Squirrels of Mediterranean origin (n = 20) were not found infected with B. burgdorferi sensu-lato (s.l.), which is almost certainly related to the low occurrence of I. ricinus in this region. Based on individuals analyzed in the other regions of France, 11.5% (n = 26) unweaned young harboured B. burgdorferi s.l., which indicates that infection occurred already in the nest. In adults (n = 227), the prevalence of infection with B. burgdorferi s.l. was 27.3%, with 18.9% B. burgdorferi s.s., 11.9% B. afzelii, and 3.5% B. garinii. The season and the body mass, sex, and geographic origin of adults had no effect on the frequency of infection. Infection prevalence of S. vulgaris is among the highest found in rodents in Europe, particularly for B. burgdorferi s.s. supporting the hypothesis that sciurids are particularly suitable hosts for this genospecies.