Technical Abstract: Little information is available concerning the presence of viable Toxoplasma gondii in tissues of goats worldwide. In the present study, the prevalence of T. gondii was determined in 234 goats from Maryland, Virginia and Pennsylvania, USA. Hearts of 234 goats were obtained from a local grocery store. Blood clot or fluid removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Antibodies to T. gondii were found in 125 (53.4%) of 234 goats, with titers of 1:5 in 20, 1:10 in 44, 1:20 in 16, 1:40 in 5, 1:160 in 5, 1:320 in 5, and 1:640 or higher in 30 goats Hearts of 112 goats (46 goats,<1:5, and 66 goats 1:10 or higher) were used for isolation of viable T. gondii by bioassay in mice. For bioassays in mice, 50 g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. Toxoplasma gondii was isolated from 29 goats; from hearts of 1 of 46 with titers of <1:5, 1 of 9 with titers of 1:10, 1 of 3 with titers of 1:40, and 26 of 40 with titers of 1:160 or higher. Three isolates were virulent to out bred Swiss Webster mice. All T. gondii isolates were subsequently grown in cell cultures. Genotyping of the 28 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) from DNA obtained from cell culture grown tachyzoites revealed 12 genotypes. Nine isolates were clonal Type II lineage, four isolates were Type II except at the Apico locus, and three isolates were clonal Type III. The remaining 12 strains were divided into 9 atypical genotypes, including four new genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in goats, which has important implications in public health. We believe this is the first genetic analysis of T. gondii isolates from goats in the USA.