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Sheep are highly susceptible to <i>Toxoplasma gondii</i>, and miscarriage is the main clinical feature. This study investigated 227 sheep samples (210 myocardial tissues from slaughterhouses, 6 ewe serum samples, 3 aborted fetuses, and 8 dead lambs from veterinary clinics) from central China for <i>T. gondii</i> infection. Antibodies against <i>T. gondii</i> were detected using the modified agglutination test (MAT). PCR was performed to detect <i>T. gondii</i> DNA in the tissue samples. The results showed that four samples were seropositive (MAT titer ≥ 1:100), with a seroprevalence of 1.8% (4/227). The seropositive samples included two myocardial samples from a slaughterhouse, one ewe and its aborted fetus from a veterinary clinic. The results revealed that 7 out of 207 (3.4%) sheep tissue samples were PCR-positive, including two myocardial tissue samples from slaughterhouses, three aborted fetuses, and two lambs from veterinary clinics. <i>Toxoplasma gondii</i> vertical transmission had occurred in two of three pairs of ewes and her pups. One viable <i>T. gondii</i> strain (TgSheepCHn14) was isolated from the myocardial tissues of sheep from a slaughterhouse. Tachyzoites were obtained from cell cultures at 70 days following seeding in the brains and lungs of mice. This strain was non-lethal to Swiss mice. The number of parasite brain cysts in mice decreased with time post-infection (<i>p</i> < 0.05). Overall, the prevalence of <i>T. gondii</i> in the sheep samples was low. Although the samples were scattered, and not from planned collections, the current study detected <i>T. gondii</i> antibodies and DNA in aborted fetuses, indicating that vertical transmission could occur and maintain the parasites in sheep herds without exogenous infection.