Tropical Bovine Theileriosis is an important tick-borne disease. This study aims to assess the occurrence ofTheileria annulatainfection in two indigenous Portuguese cattle breeds. A total of 843 blood samples collected from animals of Alentejana (n = 420) and Mertolenga (n = 423) breeds were analyzed. The detection ofTheileria annulatawas determined by amplification of a fragment of the merozoite-pyroplasm surface antigen gene with 319 base pairs (bp). The prevalence found (10.8%) is lower than that reported in previous studies (21.3%). A statistically significant difference was found for positivity between breeds (p< 0.05). There is also a higher probability of older animals being positive compared to younger ones (p< 0.05). The region where Mertolenga animals are located is shown to have a significant impact on positivity (p< 0.05). Thus, the development of sustainableT. annulatacontrol strategies and their implementation, adapted to the epidemiological conditions of higher risk, will be extremely important.