Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans. However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans.