ABSTRACTCandida albicansandCandida dubliniensisare diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains ofC. albicansof the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploidC. albicansgenerated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n+ 2n) ofC. albicansandC. dubliniensisby spheroplast fusion. Heterodiploid (n+n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicansCse4/C. dubliniensisCse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of eitherC. albicansorC. dubliniensis. In keeping with the recent discovery of the existence of haploidC. albicansstrains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny ofC. albicansorC. dubliniensisin future.