AbstractAneuploidy, an unbalanced karyotype in which one or more chromosomes are present in excess or reduced copy number, causes an array of known phenotypes including proteotoxicity, genomic instability, and slowed proliferation. However, the molecular consequences of aneuploidy are poorly understood and an unbiased investigation into aneuploid cell biology is lacking. We performed high-throughput screens for genes the deletion of which has a synthetic fitness cost in aneuploid Saccharomyces cerevisiae cells containing single extra chromosomes. This analysis identified genes that, when deleted, decrease the fitness of specific disomic strains as well as those that impair the proliferation of a broad range of aneuploidies. In one case, a chromosome-specific synthetic growth defect could be explained fully by the specific duplication of a single gene on the aneuploid chromosome, highlighting the ability of individual dosage imbalances to cause chromosome-specific phenotypes in aneuploid cells. Deletion of other genes, particularly those involved in protein transport, however, confers synthetic sickness on a broad array of aneuploid strains. Indeed, aneuploid cells, regardless of karyotype, exhibit protein secretion and cell-wall integrity defects. Thus, we were able to use this screen to identify novel cellular consequences of aneuploidy, dependent on both specific chromosome imbalances and caused by many different aneuploid karyotypes. Interestingly, the vast majority of cancer cells are highly aneuploid, so this approach could be of further use in identifying both karyotype-specific and nonspecific stresses exhibited by cancer cells as potential targets for the development of novel cancer therapeutics.