Ingesting meat from lamb or mutton containing cysts of Toxoplasma gondii can lead to human infection, defined as toxoplasmosis. Many studies worldwide reported high antibody levels to T. gondii in sheep, but this does not always indicate that tissues destined for consumption contain infectious cysts. This study aimed to understand T. gondii occurrence in sheep muscles and the correlation between seropositivity and parasite presence. Samples from 349 sheep (blood, heart and diaphragm) underwent ELISA, real-time PCR and histological testing. Despite high seroprevalence rates, T. gondii DNA was found in only 3.7% of tested sheep (13/349), all adults. Histological tests did not detect T. gondii tissue cysts in any samples tested. The results agree with previously published research and might be influenced by the uneven distribution of tissue cysts in carcasses or muscle and by the analytical methods used. Toxoplasma gondii is a zoonotic pathogen and the ingestion of tissue cysts by consumption of lamb or mutton has been identified as a possible cause of infection in humans. Many serological surveys in sheep have been performed, showing relevant serological rates; however, while the detection of antibodies indicates an exposure to T. gondii, this does not necessarily imply the presence of tissue cysts in edible tissue. The current study aims to provide further understanding on the occurrence of T. gondii in sheep muscles and the strength of correlation between serological positivity and presence of the parasite in sheep. From 349 sheep, samples (i.e., blood, heart and diaphragm) were collected and subjected to ELISA tests, real-time PCR and histological tests. Despite the high seroprevalence, T. gondii DNA was detected in the heart and/or the diaphragm from 13 out of the 349 tested sheep (3.7%); all were adults (13/191). Furthermore, the histological tests did not reveal the presence of T. gondii tissue cysts in any of the examined portions of interventricular septum. It should be considered that the likelihood of detecting genetic material of the parasite is probably influenced by the uneven distribution of the tissue cysts in the carcass as well as the methodology applied. The findings of this study support the importance of describing the uncertainty associated with the data used for risk assessment to reduce inaccurate estimation or risk overestimation.