It was generally believed that Toxoplasma gondii had a clonal population structure with three predominant lineages, namely types I, II and III. This was largely based on genotyping of more than 100 T. gondii isolates originating from a variety of human and animal sources in North America and Europe. Recent genotyping studies on T. gondii strains from wild animals or human patients from different geographical regions revealed the high frequency of non-archetypal genotypes, suggesting the overall diversity of the T. gondii population might be much higher than we thought. However, as most genotyping studies had relied on a few biallelic markers, the resolution and discriminative power of identifying parasite isolates were quite low. To date, there is no commonly used set of markers to genotype T. gondii strains and it is not feasible to compare results from different laboratories. Here, we developed nine PCR-restriction fragment length polymorphism markers with each of them capable of distinguishing the three archetypal T. gondii alleles in one restriction-enzyme reaction by agarose gel electrophoresis. Genotyping 46 T. gondii isolates from different sources using these markers showed that they could readily distinguish the archetypal from atypical types and reveal the genetic diversity of the parasites. In addition, mixed strains in samples could be easily detected by these markers. Use of these markers will facilitate the identification of T. gondii isolates in epidemiological and population genetic studies.