Genetic analysis of the SAG2 locus was performed to determine the prevalence of the main genotypes of Toxoplasma gondii (SAG2 types I, II, and III) associated with humans, cats, birds and guinea pig toxoplasmosis in Colombia. This typing was directly performed on clinical samples and autopsy material from human or animals. A total of 50 from 146 samples were positive by specific B1 Toxoplasma PCR assay and then were analyzed by restriction fragment length polymorphism in PCR-amplified SAG2 products. Characterization of the SAG2 gene was successful in 33 (66%) of the samples. Genotyping indicated that 31 (93.9%) were SAG2 type I, 1 was SAG2 type III and 1 was atypical. In birds and cats all samples were SAG2 type I. Results support a predominance of the Toxoplasma SAG2 type I circulating in human and animals in South America.