(1) Background: Toxoplasma gondii is an important zoonosis and one of the major causes of abortion in sheep worldwide. (2) Methods: We performed a 2-year longitudinal serological anti-T. gondii IgG screening on a cohort of a spatially confined population of a Portuguese autochthonous sheep breed in central Portugal. (3) Results: From the screening of the 2015 and 2016 sera, an increase of seroprevalence was observed (57.7% (95% CI: 49.9–65.3%) versus 69.1% (95% CI: 61.5–75.9), from 2015 and 2016, respectively) (p = 0.031). (4) Conclusions The present study is the first to provide prospective data on the anti-T. gondii serological status of a sheep cohort in Portugal, showing an increase in the occurrence of T. gondii. There is a need to provide a clearer understanding of T. gondii epidemiology in Portugal, ideally by implementing monitoring programs on sentinel herds, not only due to the high impact of T. gondii on animal health but also for it being a zoonosis.
