Lyme borreliosis is rapidly emerging in the United Kingdom, with over 1000 cases per annum now reported. Lyme borreliosis is caused by the Borrelia burgdorferi sensu lato (s.l.) group of spirochetes, which are transmitted by ixodid ticks. In the United Kingdom, Ixodes ricinus is recognized as the principal vector of the spirochetes, and this tick species is widely distributed across the country. However, as yet, it is unclear whether the distribution of B. burgdorferi essentially mirrors that of its vector, or if there are marked differences between the 2. The aim of this survey was to investigate the prevalence of B. burgdorferi in I. ricinus populations across northern England, north Wales, and the Scottish Border region. We surveyed for questing I. ricinus nymphs and adults at 17 sites, encountering ticks at 12. At 8 sites, large numbers (>160) ticks were collected, and at 3 of these sites B. burgdorferi infection prevalence was significantly higher (>7.5%) than the other 5 (<1.0%). Habitat type was associated with B. burgdorferi prevalence, with ticks from deciduous and mixed woodland being significantly more likely to be infected than those from other habitat types. Identification of infecting Borrelia species indicated that Borrelia valaisiana was the most common and widespread species encountered. B. garinii was common at sites where infection prevalence was high, and B. afzelii was also occasionally encountered at these sites. The survey revealed a surprisingly uneven distribution of B. burgdorferi s.l. across the region, thereby indicating that the presence of ticks does not necessarily mean the presence of the pathogen. Indeed, the spirochete appears to be absent or very rare at some sites where ticks are abundant.