Isolates of Toxoplasma gondii, which is responsible for a wide range of clinical manifestations are grouped into three clonal lineages of different virulence in mice. However, it is not clear whether this genotypic pattern is associated with the clinical profile of the disease in humans nor is the geographical distribution of the genotypes known. This is mainly due to difficulties in obtaining parasitic DNA from patients. The available data are therefore limited and originate from acute or congenital infections or from animals. A non-invasive assay is needed to address issues of strain type, geographical distribution and severity of clinical toxoplasmosis. To serotype T. gondii strains, we have developed an enzyme-linked immunosorbent assay (ELISA) that uses polymorphic polypeptides specific to the three clonal lineages and derived from two dense granule antigens, GRA5 and GRA6. Two hundred and fifty-two sera from chronically infected pregnant women from three different European countries and Colombia were investigated. The analysis of genotype-specific antibody response showed a homogeneous type II distribution in the European samples compared with types I and III but no type II in the Colombian population. Our data concord with those obtained from the genotyping of other isolates from Europe and South America. We demonstrated that, despite some limitation due to antigen and/or antibody specificity, serotyping is a promising assay to investigate the relationship between type of strain and severity of the disease.