Piroplasmosis, a tick-borne protozoal disease, is an important disease affecting domestic and wild animals. We performed PCR-based reverse line blot hybridization (RLB) assays on blood samples obtained from 133 cattle exposed to ticks in field conditions in Minorca (Balearic Islands, Spain) in three different seasons. The oligonucleotides used were those for Theileria annulata, T. buffeli, T. taurotragi, T. velifera, Babesia bigemina, B. bovis, B. divergens, and B. major. The RLB technique allowed the simultaneous identification of T. annulata, T. buffeli, B. bigemina, and B. bovis as the piroplasms present in cattle in Minorca. Of the 133 animals, only 4 were not infected by any of the studied parasites. The results indicated endemic piroplasm infection in cattle in Minorca; especially important was the presence of T. annulata. The RLB was highly sensitive and allowed the simultaneous detection and identification of the Theileria and Babesia species in carrier cattle, which cannot be achieved by classical identification methods.