Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone acetylation and deacetylation also play a role during infection. Beginning with the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, we have identified and characterized five sirtuins encoded in the C. neoformans genome. While only two of the five deletion strains revealed mutant phenotypes in vitro, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4. The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene, suggesting lost epigenetic memory could not be restored in this class of mutant and highlighting regulation of histone modification as a means to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans. Remarkably, we have been acquired support of this model from an unexpected source. Analysis of a collection of type strain H99 subcultures from the Cryptococcus research community revealed that the most commonly used strains belong to a highly compromised hypervirulent and pleiotropic mutant lineage lacking a component of the SAGA actyltransferase complex. Like with sir2∆, hst3∆ and hst4∆, reintroduction of the wild-type gene does not restore the mutant’s lost epigenetic memory. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.