A serosurvey was performed to assess exposure to Toxoplasma gondii in zoo animals in Spain and to determine the dynamics of seropositivity in some zoo species over time. Sera from 393 zoo animals belonging to 91 species were collected in eight zoos in Spain between 2007 and 2019. Additionally, 39 of the 393 animals from five of the analyzed zoos were longitudinally sampled during the same study period. Antibodies to T. gondii were detected in 165 (42.0 %; CI95 %: 37.1–46.9) of 393 animals by the modified agglutination test (MAT, cut-off = 25). Antibody titers of 25, 50, 100 and ≥500 were found in 25 (15.2 %), 65 (39.4 %), 26 (15.8 %), and 49 (29.7 %) of the seropositive animals, respectively. Feeding habits (carnivorous species) was a risk factor (OR = 5.6; P < 0.001; CI95 %: 3.8–9.0) potentially associated with T. gondii exposure. Eighteen (46.2 %) of the 39 animals longitudinally sampled were always seropositive and five animals (12.8 %) (two spotted hyaena [Crocuta crocuta], one blesbok [Damaliscus pygargus], one white rhinoceros [Ceratotherium simum] and one mouflon [Ovis aries musimon]) seroconverted during the study period. Our results evince a wide circulation of T. gondii in zoos in Spain, including wild felids. Zoo animals can be useful sentinel species to monitor T. gondii circulation in epidemiological scenarios with a close human-wildlife interface. Control measures should be implemented in zoo parks to minimize the risk of exposure of zoo species to T. gondii.