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Toxoplasma gondii is a protozoan parasite with high zoonotic potential. Currently, no information is available on natural toxoplasmosis in domestic rabbits in Romania; therefore, the aim of the present study was to evaluate the seroprevalence of T. gondii IgG antibodies and to determine the prevalence of T. gondii DNA in domestic rabbits in our country. In total, 372 domestic rabbits were tested. Blood samples were obtained from 352 animals and tissue samples were obtained from 49 animals, of which 29 animals provided paired blood–tissue samples. Samples were collected from pet rabbits and from rabbits raised in household settings, hereafter referred to as farm rabbits. Sera samples were analyzed using a modified agglutination test (MAT), with a cut-off of 1:24, for anti-T. gondii antibody IgG-type detection, and the tissue specimens were tested by nested polymerase chain reaction (nested PCR) targeting the B1 gene for T. gondii DNA detection. A seropositivity of 16.5% (58/352) was obtained by MAT, while the prevalence of T. gondii DNA was 4.1% (2/49). The present study highlighted the presence of T. gondii in domestic rabbits in Romania, which suggests that rabbit meat consumption may represent a potential risk to human health and therefore warrants further attention. Moreover, to the best of our knowledge, this is the first study to report data on the prevalence of T. gondii in domestic rabbits from Romania.