Bovine borreliosis, caused by Borrelia theileri which is transmitted via hard tick bites, is associated with mild clinical symptoms, such as fever, lethargy, hemoglobinuria, anorexia, and anemia. Borrelia theileri infects various animals, such as cattle, deer, horses, goats, sheep, and wild ruminants, in Africa, Australia, and South America. Notably, no case of B. theileri infection has been reported in Korean cattle to date. In this study, 101 blood samples were collected from a Korean indigenous cattle breed, among which 1.98% tested positive for B. theileri via nested PCR. The obtained sequences exhibited high homology with B. theileri strains identified in other regions. Phylogenetic analysis of 16S rRNA confirmed the B. theileri group affiliation; however, flagellin B sequences exhibited divergence, potentially due to regional evolutionary differences. This study provides the first molecular confirmation of B. theileri infection in Korean livestock. Further isolation and nucleotide sequence analyses are necessary to better understand the presence of B. theileri strains in cows in Korea.