Genomic DNAs from 35 Toxoplasma gondii strains were amplified by random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) using 18 arbitrary 10-mer primers. At least four primers were found to generate DNA fragments that discriminate the 35 T. gondii strains into a genotype of virulent strains and a genotype of avirulent strains. Primer B12 was found to generate a virulence-specific fragment and primers B5, C8, and C20 were found to generate avirulence-specific fragments, which in all cases clearly identified either the virulence phenotype or the avirulence phenotype, respectively. In addition, the DNA polymorphic bands detected were analyzed by parsimony and distance analysis. A similar genetic relationship among the T. gondii strains was determined by the two phylogenetic methods, which use completely different assumptions. Consistent with the division of the 35 strains into a genotype of virulent strains and a genotype of avirulent strains, both analyses revealed 2 clonal lineages directly correlated with murine virulence. These results strongly support the hypothesis that the genus Toxoplasma may actually contain two clonal lineages correlated with virulence, which have evolved independently following their initial separation.