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Questing Ixodes ricinus ticks were collected from six locations throughout Ireland and 638 nymphs, 111 females and 118 males were investigated for infection with Borrelia burgdorferi sensulato (s.l.). The total prevalence of B. burgdorferi s.l. in the ticks was determined as 14.9% by polymerase chain reaction (PCR) amplification of the spacer region of 5S-23S rRNA genes. Infection prevalence was significantly higher in adult (20.1%) Ixodes ricinus compared to nymphs (13.1%). The prevalence of infection in adult male and female ticks was similar (19.5% and 20.7% respectively). The genomospecies B. burgdorferi sensu stricto, B. afzelii, B. garinii and group VS116 were identified by reverse line blot (RLB) using genomospecies specific oligonucleotide probes. The most prevalent B. burgdorferi genomospecies identified were VS116 (34.6%), B. garinii (24.3%) and B. burgdorferi sensu stricto (18.4%). B. afzelii was uncommon (6.6%). Multiple infections were observed in 13.2% of the infected ticks. The distribution of the genomospecies showed geographical variation and also seemed to be influenced by the nature of the habitat. A broad range of genomospecies seemed to be associated with the presence of a wide spectrum of potential reservoir hosts in the habitat and also with a high overall prevalence of B. burgdorferi s.l.