Lyme borreliosis is a vector-borne disease transmitted through the bite of ticks infected by Borrelia burgdorferi sensu lato group, including B. burgdorferi sensu stricto, B. afzelii, and B. garinii. The goal of the present study was to detect Borrelia species in ticks infesting wild Korean water deer (KWD; Hydropotes inermis Swinhoe), using molecular and genotypic analyses. In total, 48 ticks were collected from KWD, all of which were morphologically identified as Haemaphysalis longicornis Neumann that is dominant in Korea. Nested PCR was performed to detect the Borrelia-specific 5S (rrf)–23S (rrl) intergenic spacer region and the outer surface protein A (ospA) genes in ticks. Both rrf–rrl and ospA were amplified from one of the 48 ticks (2.1%) and were identified as B. afzelii. To our knowledge, this study constitutes the first molecular detection of B. afzelii in Haemaphysalis ticks in Korea. Because B. afzelii is a zoonotic tick-borne pathogen, understanding the molecular characteristics of this bacterium is important for preventing the transmission of Borrelia from ticks to other animals and humans.