Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans, to eliminate the additional mutations present in the ATCC 24067A genetic background, demonstrated that the hypermutator phenotype of the pol3D270G mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.