Babesia ovata and Babesia major are two newly identified large Babesia species infective to cattle in China. There is a demand for specific tools for discrimination between the two species due to the confusion of their classification based on traditionally classification methods, such as tick vector, morphology, and pathogenicity. In this study, the internal transcribed spacers (ITS including ITS1, 5.8S coding region and ITS2) were originated from four isolates of B. ovata and one of B. major from different geographic regions of China, and a phylogenetic tree was inferred. It was demonstrated that all of the four isolates of B. ovata were grouped into one cluster, while B. major isolate was placed in another. The sequence percent identity showed that B. ovata isolates had the minimum 85.5% identity, whereas B. major showed only the maximum 46.6% identity to the four B. ovata isolates. In addition, the identity of ITS1 and ITS2 of these Babesia isolates was discussed. The findings implied that the four B. ovata isolates have a quite close relationship, whereas the B. major isolate showed far relationship with all of these B. ovata isolates. This finding may lead to the conclusion that there is actual existence of two large Babesia infective to cattle in China, one is B. ovata and the another is B. major.