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This study investigated the differences in genome structure between haploid serotype A and D isolates and AD-hybrid strains of Cryptococcus neoformans, and the correlation between the karyotype of A and D strains with their mating ability. The electrophoretic karyotyping of 16 AD-hybrid, eight haploid serotype-A MATα, and eight haploid serotype-D MATαC. neoformans isolates was performed. These 32 isolates presented, two by two, the same genotype and flow cytometry profile. Five clusters were identified, each including VNI (serotype A), VNIV (serotype D) haploid strains and VNIII AD hybrids. Similarly, mating types were also randomly distributed in the five clusters. In addition, AD-hybrid isolates, with double content of DNA, showed only a slight increase in both the number of chromosomal bands and the calculated genome size compared with haploid isolates. Data support the hypothesis that hybrid isolates are aneuploids (2n+x) rather than eudiploids (2n). In addition, a set of six mating type a strains were karyotyped and then used for mating experiments carried out crossing the haploid isolates with similar or different karyotype profile strains. Isolates with completely different karyotype were able to mate confirming that meiosis occurred even in the presence of chromosomes of different lengths.