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 
accord-ing 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.