Babesia orientalis is the causative agent of babesiosis in water buffalo (Bubalus babalis, Linnaeus, 1758). In this study, a TaqMan real-time PCR assay was developed for quantitative detection of B. orientalis in water buffalo. Hybridization probe and oligonucleotide primers were designed based on the v4 region of 18S rRNA gene. Detection limit was determined at 2 parasites. Blood samples were collected from experimentally infected water buffalo, as well as from 180 field samples, which were collected from 4 different geographical locations to the north and south of the Yangtse River. The parasite was detected by real-time PCR on day 2 until day 39 post-infection, while reverse line blot (RLB) was on day 6 until day 36 in experimentally infected water buffalo. For the results of 180 field samples, statistical analysis showed no significant difference in relative effectiveness of real-time PCR and RLB. The analysis also indicated that there was no difference in the prevalence of B. orientalis between the regions of south and north of the Yangtse River by both the real-time PCR assay and RLB detection. These results indicated that the parasite infection has spread to the north of the Yangtse River.