Technical Abstract: Most T. gondii strains in North America and Europe belong to three archetypal clonal lineages including the Type I, II and III but, isolates from Brazil are highly diverse. Here, we analyzed 164 T. gondii isolates from three countries in Central America (Guatemala, Nicaragua, Costa Rica), from one country in Caribbean (Grenada) and five countries from South America (Venezuela, Colombia, Peru, Chile, Argentina). The multilocous polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based genotyping of 11 polymorphic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, L358, PK1, C22-8, C29-2, Apico) were applied to 148 free-range chickens (Gallus domesticus) isolates and 16 isolates from domestic cats (Felis catus) in Colombia; 42 genotypes were identified. Linkage disequilibrium analysis indicated more frequent genetic recombination in populations of Nicaragua and Colombia, and to a lesser degree in populations of Costa Rica and Argentina. Bayesian structural analysis identified at least three genetic clusters, and phylogenetic network analysis identified four major groups. All three clonal Type I, II and III lineages were identified from Central and South America, with a high frequency of the Type III lineage. Based on the 11 RFLP markers, closely related strains to the clonal Type I and III lineages were readily identified from Central and South America populations, suggesting Type I and III lineages might have originated from this geographical region. The clear dominance of the Type II strains in Chile , and its absence from the rest of populations suggest that Type II lineage might have been recently imported to South America from northern hemisphere. Taken together, this study revealed highly diverse T. gondii populations in Central and South America, and suggested different origins of the three clonal Type I, II and III lineages.