Theileria orientalis is the causative agent of benign theileriosis, which is distributed mainly in Asian countries, and causes serious economic losses in the livestock industry. The present study was performed to investigate the epidemiology of T. orientalis infections in cattle in the Republic of Korea (ROK) and to characterize the genetic diversity of T. orientalis based on the major piroplasm surface protein (MPSP) genes. In 2015, between July and August, blood samples were collected from 138 asymptomatic cattle in four different geographical regions (Hoengseong, Jeongeup, Namwon, and Jeju island) in the ROK. In total, 57 blood samples (41.3 %, 57/138) were positive for T. orientalis based on PCR amplification of the MPSP gene. A high prevalence of T. orientalis infection was observed in Jeju island, whereas the infection rate was relatively low in Jeongeup. Phylogenetic analysis showed that isolates identified in this study belonged to four MPSP genotypes, specifically types 1, 2, 3, and 7. The distribution of the four genotypes varied considerably among the four regions; types 1, 2, and 3 were detected in Jeju island, whereas types 1 and 7 were found in Namwon, types 1 and 2 in Jeongeup, and type 2 in Hoengseong. To our knowledge, this is the first report on the identification of type 7 T. orientalis in cattle in the ROK. These results suggest that the MPSP genotypes detected in this study showed genetic diversity related to geographical location. Our findings revealed that the T. orientalis infection rate was relatively high, indicating that T. orientalis infection is closely associated with grazing. Of the four MPSP genotypes, the prevalence of the most pathogenic type 2 was relatively high in the ROK. Therefore, further studies should focus on the development of an effective monitoring and prevention program for T. orientalis.