Abstract: Centromeres are specialized structures on eukaryotic chromosomes that couple chromosome movements to spindle microtubule movements and allow accurate chromosome segregation during cell division. In spite of these vital functions, recent evidence strongly suggests that epigenetic regulation rather than the primary DNA sequence of the centromere plays a dominant role in the specification of centromeres. The key determinant of centromere identity is the centromere-specific histone H3 variant CENP-A (also known as CenH3). This review highlights exciting new findings examining the mechanism of centromere specification in distinct yeast species, which provides insights into CENP-A nucleosome assembly and establishment of centromere identity.