The species complexesCryptococcus neoformansandCryptococcus gattiiare the causative agents of cryptococcosis. Virulence and susceptibility to antifungals may vary within each species according to the fungal genotype. Therefore, specific and easily accessible molecular markers are required to distinguish cryptic species and/or genotypes. Group I introns are potential markers for this purpose because they are polymorphic concerning their presence and sequence. Therefore, in this study, we evaluated the presence of group I introns in the mitochondrial genescobandcox1in differentCryptococcusisolates. Additionally, the origin, distribution, and evolution of these introns were investigated by phylogenetic analyses, including previously sequenced introns for themtLSUgene. Approximately 80.5% of the 36 sequenced introns presented homing endonucleases, and phylogenetic analyses revealed that introns occupying the same insertion site form monophyletic clades. This suggests that they likely share a common ancestor that invaded the site prior to species divergence. There was only one case of heterologous invasion, probably through horizontal transfer toC. decagattii(VGIV genotype) from another fungal species. Our results showed that theC. neoformanscomplex has fewer introns compared toC. gattii.Additionally, there is significant polymorphism in the presence and size of these elements, both among and within genotypes. As a result, it is impossible to differentiate the cryptic species using a single intron. However, it was possible to differentiate among genotypes within each species complex, by combining PCRs ofmtLSUandcox1introns, forC. neoformansspecies, andmtLSUandcobintrons forC. gattiispecies.