Lyme borreliosis is one of the most prevalent tick-borne infectious zoonotic diseases caused by spirochetes of the sensu lato group. The present study assessed the risk factors and prevalence of Lyme borreliosis in ticks parasitizing domestic and wild animals. A total of 589 ticks (329 tick pools) collected from animals were identified as , (85.7%), (10.0%), and (4.3%) using morphological and molecular methods in South Korea. In this study, the 5S-23S gene sequences of (6/329, 1.8%) were detected in ticks taken from mammals, including ticks from horses (2/147 pools, 1.4%), wild boar (1/19 pools, 5.3%), native Korean goats (NKG, 2/34 pools, 5.9%), and Korean water deer (1/129 pools, 0.8%). Unfortunately, , , and genes were not able to be amplified in the present study. To our knowledge, our results are the first inclusive data available for circulation in several tick species taken from NKG, horses, and wild boar in South Korea. We believe that the current findings extend our knowledge of the distribution and possible vector spectrum of spp. We recommend continuous evaluation of the potential public health threat posed by infected ticks.