A PCR-based survey of hemoprotozoa parasites detected Babesia bigemina, Theileria orientalis and Trypanosoma theileri among cattle and water buffalo in Vietnam, and a new Babesia sp. closely related to Babesia ovata was detected in cattle only. In addition, Theileria annulata and Trypanosoma evansi were not detected in both cattle and water buffalo. Phylogenetic analysis detected T. orientalis MPSP genotypes 3, 5, 7 and N3 in cattle and 5, 7, N1 and N2 in water buffalo. Additionally, water buffalo-derived T. theileri CATL sequences clustered together with a previously reported cattle-derived sequence from Vietnam. This is the first report of a new Babesia sp. in cattle, and T. orientalis MPSP genotype 7 and T. theileri in water buffalo in Vietnam.