The prevalence of Borrelia species infection was determined by polymerase chain reaction (PCR) in 138 ticks collected from dogs which were walked regularly in the wooded areas near the city of Eindhoven, the Netherlands. The PCR amplified the spacer region between the 5S and 23 S rRNA genes, and the Borrelia species was identified by hybridization with specific probes. Borrelia burgdorferi sensu lato was present in 20 of 138 (14.5%) ticks. Four species were identified: B. burgdorferi sensu stricto (n = 8), B. afzelii (n = 4), B. garinii (n = 2), and B. valaisiana (n = 2). One PCR product was non-typeable. Three ticks contained more than one species, all including B. burgdoferi sensu stricto, and one tick even contained four species. There was a significant difference (P < 0.05) in prevalence of B. burgdorferi sensu stricto between non-engorged ticks (either questing or attached) and semi-engorged ticks, 12% (10 of 85) and 2% (1 of 53), respectively.