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Background: This study investigated Borrelia species prevalence in ticks from vegetation, through a molecular method, in Gwangju Metropolitan City, South Korea. Methodology/Principal findings: A total of 484 ticks were collected through flagging and dragging in a suburban area of Gwangju Metropolitan City, South Korea, in 2014. These ticks were morphologically identified and subjected to nested PCR, targeting Borrelia–specific CTP synthase (pyrG), outer surface protein A (ospA) and flagellin (flaB) genes. Molecular biological species identification of Borrelia-positive ticks was conducted via 16S rRNA PCR assays. Of the 484 ticks collected, 417 (86.2%) were identified as Haemaphysalis longicornis, 42 (8.7%) as H. flava, and 25 (5.2%) as Ixodes nipponensis. All the ixodid ticks containing Borrelia species bacteria were confirmed to be I. nipponensis adults, by both morphological and molecular methods. Of the 25 I. nipponensis ticks collected, four (16%) were positive for Borrelia species, three of which were B. afzelii and one B. miyamotoi. Conclusions/Significance: Our study has shown the harboring of B. miyamotoi by I. nipponensis in South Korea. Morphological and molecular genetic analyses revealed that, in South Korea, I. nipponensis could potentially transmit B. miyamotoi to humans. [ABSTRACT FROM AUTHOR]