The study of quantitative traits provides a window on the interactions between multiple unlinked genetic loci. The interaction between hosts and pathogenic microbes, such as fungi, involves aspects of quantitative genetics for both partners in this dynamic equilibrium. One important pathogenic fungus is Cryptococcus neoformans, a basidiomycete yeast that can infect the human brain and whose mating system has two mating type alleles, a and α. The α mating-type allele has previously been linked to increased virulence potential. Here congenic C. neoformans strains were generated in the two well-characterized genetic backgrounds B3501α and NIH433a to examine the potential influence of genes outside of the mating-type locus on the virulence potential of mating type. The congenic nature of these new strain pairs was established by karyotyping, amplified fragment length polymorphism genotyping, and whole-genome molecular allele mapping (congenicity mapping). Virulence studies revealed that virulence was equivalent between the B3501 a and α congenic strains but the a strain was more virulent than its a counterpart in the NIH433 genetic background. These results demonstrate that genomic regions outside the mating type locus contribute to differences in virulence between a and α cells. The congenic strains described here provide a foundation upon which to elucidate at genetic and molecular levels how mating-type and other unlinked loci interact to enable microbial pathogenesis.