Piroplasmosis caused by tick-borne hemoprotozoans of several Theileria and Babesia species has a major impact on livestock production worldwide. A reverse line blotting assay that includes genus- and species-specific probes for Theileria and Babesia species was used to assess the occurrence of these parasites in blood samples collected from 1407 healthy bovines throughout mainland Portugal. The global prevalence of piroplasm-infected animals was 36.8%, although significant differences were found between various regions. Higher prevalence was found in the southern regions (42.4% in Lisbon and Tagus Valley, 51.6% in Alentejo, and 40.0% in Algarve) compared to central (23.1%) and northern (12.8%) Portugal. Theileria annulata was the most frequently found species with a prevalence of 21.3%. The prevalence values of this pathogenic species were higher in the southern regions. The second most prevalent species was T. buffeli that infected 10.1% of the bovines. A low prevalence was found for Babesia infections (7.9%) with B. bigemina as the most frequent species. Some animals were infected with T. ovis and B. occultans. Veterinary practitioners and stakeholders should be made aware of the existence of a relatively high prevalence of carrier animals infected with T. annulata in Portugal, given the potential threat this pathogenic parasite represents to the cattle industry.