Strains of Toxoplasma gondii can be grouped into three predominant clonal lineages with members of the type I group being uniformly lethal in mice. To elucidate the basis of this extreme virulence, a genetic cross was performed between a highly virulent type I strain (GT-1) and a less-virulent type III strain (CTG), and the phenotypes of resulting progeny were analyzed by genetic linkage mapping. Analysis of independent recombinant progeny identified several quantitative trait loci that contributed to acute virulence. A major quantitative trait locus located on chromosome VII accounted for ≈50% of the virulence phenotype, whereas a minor locus on chromosome IV, linked to the ROP1 gene, accounted for ≈10%. These loci are conserved in other type I strains, indicating that acute virulence is controlled by discrete genes common to the type I lineage.