To explore the tick distribution and prevalence of Borrelia in Zhejiang Province, we performed a survey in nine sites. A total of 447 adult ticks of 11 species were captured and the dominant tick species were Haemaphysalis longicornis and Ixodes sinensis and the abundance of tick species in different areas varied significantly. Overall, 4.70% of the ticks were polymerase chain reaction (PCR) positive for Borrelia. The average PCR positive rates were 5.19% for H. longicornis, 3.45% for Amblyomma testudinarium, 1.06% for I. sinensis, 5.00% for Rhipicephalus (Boophilus) microplus, and 19.44% for Ixodes granulatus, respectively. No Borrelia DNA was detected in Rhiphicephalus haemaphysaloides, Haemaphysalis yeni, Dermacentor taiwanensis, Haemaphysalis hystricis, Hyalomna asiaticum, and Ixodes ovatus. The prevalence of Borrelia was significantly different among tick species and the prevalence in I. granulatus was significantly higher than that in other tick species. Of note, experimentally confirmed vectors for B. burgdorferi s.l. including I. sinensis and I. granulatus were found in Zhejiang Province. Two species of B. burgdorferi s.l. exist in Zhejiang Province of which 12 sequences were most similar to the sequence of Borrelia garinii and nine sequences were most similar to the sequence of Borrelia valaisiana or Borrelia yangtze sp. nov.