| Summary: | (1) Background: <i>Toxoplasma gondii</i> 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-<i>T. gondii</i> 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) (<i>p</i> = 0.031). (4) Conclusions The present study is the first to provide prospective data on the anti-<i>T. gondii</i> serological status of a sheep cohort in Portugal, showing an increase in the occurrence of <i>T. gondii</i>. There is a need to provide a clearer understanding of <i>T. gondii</i> epidemiology in Portugal, ideally by implementing monitoring programs on sentinel herds, not only due to the high impact of <i>T. gondii</i> on animal health but also for it being a zoonosis.
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