Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, α strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and α strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and α strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and α congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and α strains are equivalent in peripheral tissues but α cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and α strains in the most common pathogenic variety and suggest an explanation for the prevalence of α strains in clinical isolates.