The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of mortality among the AIDS population and is known for frequently causing life-threatening relapses. To investigate the potential contribution of in-host microevolution to persistence and relapse, we have analyzed several sets of serial isolates obtained from patients with acquired immunodeficiency syndrome who suffered initial and relapse episodes of cryptococcal meningoencephalitis. Despite being identical by multilocus sequence typing, these isolates differ phenotypically, exhibiting changes in key virulence factors such as nutrient acquisition, metabolic profiles, or the ability to disseminate in an animal model. Whole-genome sequencing has begun to provide insight into the nature of these strains, revealing that those characterized so far exhibit a clonal relationship, but with a few key differences. In one series we have shown the relapse isolate to have lost a predicted AT-rich interaction domain protein due to a frameshift mutation. Gene deletion of the predicted transcriptional regulator produced changes in melanin, capsule, carbon source use, and dissemination in the host, consistent with the phenotype of the relapse isolate. In addition, the deletion mutant displayed altered virulence in a murine model of infection. The observed differences suggest the relapse isolate evolved subsequent to penetration of the central nervous system and may have gained dominance following the administration of antifungal therapy. Remarkably, we have also shown this same gene to have been lost in a relapse isolate from a second patient. These data reveal the first molecular insights into how the Cryptococcus neoformans genome changes during infection of humans and the manner in which microevolution progresses in this deadly fungal pathogen.