Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To
characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and
compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic
regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages.
Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four
lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress,
mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to
be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the
other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV,
which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of
population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial
recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein
aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive
selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex.