This study was performed to investigate the prevalence of theileriosis and to compare the prevalence of this disease in Korean native cattle reared under different environmental conditions, namely, in a grazing area and a non-grazing area by polymerase chain reaction. Three hundred and one Korean native cattle (276 cows and 25 bulls) that had not received prior treatment or been vaccinated to prevent theileriosis were examined by PCR for Theileria sergenti infection from 2001 to 2002. In our study, the parasitemia range in T. sergenti-positive cattle by microscopy were from 0.1 to 3% (mean 0.8%). In terms of mean prevalence, 204 of the 301 Korean native cattle (67.8%) were positive reaction by PCR. Our results also revealed that the infection rate among cows (70.3%) was significantly higher than that among bulls (40.0%) (p < 0.01). T. sergenti infection among the over 3 year-old-group (75%) had a significant higher prevalence than that among the less than 3 year-old-group (61.8%) (p < 0.05). Our data also showed that grazing areas (76.1%) had the significant higher prevalence than non-grazing areas (51%) (p < 0.001). In conclusion, this study demonstrates that the prevalence of T. sergenti infection is high and that its prevalence in grazing cattle is higher than that in non-grazing cattle. Therefore, life-long treatment and the development of an optimal vaccine are needed to reduce the numbers of bovine theileriosis in both grazing and non-grazing areas.