Large-scale transitions in genome size from tetraploid to diploid were observed during a previous 1800-generation evolution experiment in Saccharomyces cerevisiae. Whether the transitions occurred via a one-step process (tetraploid to diploid) or through multiple steps (through ploidy intermediates) remained unclear. To provide insight into the mechanism involved, we investigated whether triploid-sized cells sampled from the previous experiment could also undergo ploidy loss. A batch culture experiment was conducted for approximately 200 generations, starting from four triploid-sized colonies and one contemporaneous tetraploid-sized colony. Ploidy reduction towards diploidy was observed in both triploid and tetraploid lines. Comparative genomic hybridization indicated the presence of aneuploidy in both the founder and the evolved colonies. The specific aneuploidies involved suggest that chromosome loss was not haphazard but that nearly full sets of chromosomes were lost at once, with some additional chromosome mis-segregation events. These results suggest the existence of a mitotic mechanism allowing the elimination of an entire set of chromosomes in S. cerevisiae, thereby reducing the ploidy level.