A molecular epidemiological survey was conducted to investigate the presence of pathogenic Borrelia burgdorferi sensu lato (s.l.) species in the forest areas of Zhejiang province, south-east China. A total of 182 ticks of 6 species and 200 rodents of 8 species were collected and individually examined for the presence of B. burgdorferi s.l. DNA by nested PCR targeting the 5S-23S rRNA intergenic spacer. Forty-one ticks of four species, Haemaphysalis concinna, Haemaphysalis longicornis, Rhipicephalus microplus and Haemaphysalis warburconi, were infected with B. burgdorferi s.l., with an overall infection rate of 23 %. Sixteen rodents of four species, Nivivener confucianus, Nivivener coxingi, Apodemus sylvaticus and Rattus losea, were positive for B. burgdorferi s.l., with an overall prevalence of 8 %. MseI RFLP analysis and sequence analysis of the positive PCR products showed that Borrelia spirochaetes in specimens consisted of Borrelia garinii, Borrelia afzelii and Borrelia valaisiana-related group. Forty (98 %) of the B. burgdorferi s.l.-positive ticks were infected with B. garinii and one (2 %) was infected with B. afzelii. Twelve (75 %) of the positive rodents were infected with B. garinii and four (25 %) were infected with the Borrelia spirochaete belonging to B. valaisiana-related group.