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Characterisation at the species level of 142 Borrelia isolates obtained from ticks, humans and rodents in Western Europe was carried out and their geographical distribution was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human samples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSF. B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vegetation and from I. ricinus engorged on birds.