Centromeric localization of Cse4 (CENP-A in humans) is essential for faithful chromosome segregation. Overexpression and mislocalization of Cse4 and its homologs leads to chromosome loss in yeast, flies and human cells. Overexpression of CENP-A is observed in cancers, howeverthe mechanisms that prevent mislocalization of CENP-A are not fully understood. Here, we used genome-wide Synthetic Genetic Array (SGA) and identified gene deletions (HIR1, HIR2, HIR3, HPC2) encoding the replication-independent histone chaperone (HIR) complex and a Cse4 E3 ubiquitin ligase, Psh1, as displaying the highest level of lethality when Cse4 is overexpressed. We determined that Hir2 interacts with Cse4 in vivo and hir2? strain shows defects in Cse4 proteolysis, mislocalization of Cse4 to non-centromeric regions and increased chromosome loss. Hir2 regulates Psh1-mediated proteolysis of Cse4 as the stability of Cse4 in psh1? hir2? strain is similar to that in the hir2? strain, the interaction of Psh1 with Cse4 is reduced in hir2? strain, the mislocalization pattern of Cse4 is similar in hir2? and psh1? strains and suppression of Cse4 proteolysis defects in hir2? strains by overexpression of PSH1. In summary, our genome-wide screen and results with HIR complex provides insights into pathways that prevent mislocalization of Cse4 thereby promoting genome stability.