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<i>Toxoplasma gondii</i>, a major zoonotic pathogen distributed worldwide, causes severe infections in humans, animals, and birds. However, limited information is available regarding <i>T. gondii</i> infection in livestock in the Republic of Korea (ROK). Herein, we determined the prevalence of <i>T. gondii</i> infection in livestock in the ROK and identified animal species that can potentially transmit <i>T. gondii</i> to humans. <i>B1</i> gene-targeting nested polymerase chain reaction detected <i>T. gondii</i> DNA in 3.3% (2/61), 2.9% (3/105), 14.1% (11/78), and 15.4% (14/91) of dairy cattle, beef cattle, Boer goats, and Korean native goats, respectively. The prevalence of <i>T. gondii</i> was significantly higher (<i>p</i> = 0.002) in goats than in cattle. The risk of contracting <i>T. gondii</i> infection was significantly higher by 6.18-fold in Korean native goats (95% confidence interval [CI]: 1.72–22.27%, <i>p</i> = 0.005) and by 5.58-fold in Boer goats (95% CI: 1.50–20.76%, <i>p</i> = 0.010) than in beef cattle. Our <i>T. gondii</i> DNA sequences exhibited 97.1–100% homology with those obtained from various hosts in other countries. To the best of our knowledge, this is the first study to report <i>T. gondii</i> infection using the blood samples of domestic ruminants in the ROK. The results revealed that the prevalence of <i>T. gondii</i> infection is higher in goats than in cattle as determined by molecular detection. Thus, these findings suggest that <i>T. gondii</i> can be transmitted from ruminants to humans via meat consumption.