Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host specific strains. This study examined the role of grey squirrels (Sciurus carolinensis), (n=679), an invasive species in the United Kingdom (UK), as B. burgdorferi s.l. hosts. We found that grey squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi s.l. in the UK, and 11.9% were infected with B. burgdorferi s.l. All four genospecies which occur in the UK were detected in grey squirrels, and unexpectedly, the bird associated genospecies B. garinii, was most common. The second most frequent infection was with B. afzelli. Genotyping of B. garinii and B. afzelli produced no evidence for strains associated with grey squirrels. Generalised linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi s.l. infection in grey squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive grey squirrels appear to become infected with locally circulating strains of B. burgdorferi s.l., further studies are required to determine their role in community disease dynamics. Our findings highlight that the role of introduced host species in B. burgdorferi s.l epidemiology can be highly variable and thus difficult to predict.