This pilot study aimed to investigate the effects of regional and seasonal variations on the prevalence of Theileria orientalis and the hematological profile of non-grazed dairy cows in Korea. A total of 365 clinically healthy lactating Holstein Friesian cows from 26 dairy farms in 7 provinces that were categorized into northern, central, and southern regions were sampled during the warm period from July to August and the cold period from October to December. The detection of T. orientalis major piroplasm surface protein gene and the hematology non-grazed dairy cows were analyzed using peripheral blood samples. The T. orientalis prevalence was 20.0% (73/365). The prevalence in the southern region was 35.9%, which was significantly higher than that in the central (21.6%) and northern (12.9%) regions (P < 0.05). The prevalence during warm period was higher (43.0%) than that during the cold season (13.5%). The infected cows showed significantly lower erythrocyte counts in the southern region (5.8 ± 0.6 M/µl) and during the warm period (5.8 ± 0.7 M/µl) compared with those in the central and northern regions and during the cold season, which affected the extended RBC parameters, including hematocrit and hemoglobin concentrations. Our findings revealed the prevalence of T. orientalis in Korea, highlighting its high occurrence during warm periods and in certain geographical regions. Climatic factors could contribute to the health and productivity of cattle, as evidenced by the prevalence of T. orientalis and its negative impact on animals.