The histone H3 variant CENP-A confers epigenetic identity to the centromere and plays crucial roles in the assembly and function of the kinetochore, thus ensuring proper segregation of our chromosomes. CENP-A containing nucleosomes exhibit unique structural specificities and lack the complex profile of gene expression-associated histone posttranslational modifications found in canonical histone H3 and the H3.3 variant. CENP-A mislocalization into noncentromeric regions resulting from its overexpression leads to chromosomal segregation aberrations and genome instability. Overexpression of CENP-A is a feature ofmany cancers and is associated with malignant progression and poor outcome. The recent years have seen impressive progress in our understanding of the mechanisms that orchestrate CENP-A deposition at native centromeres and ectopic loci. They have witnessed the description of novel, heterotypic CENP-A/H3.3 nucleosome particles and the exploration of the phenotypes associated with the deregulation of CENP-A and its chaperones in tumor cells. Here, we review the structural specificities of CENP-A nucleosomes, the epigenetic features that characterize the centrochromatin and the mechanisms and factors that orchestrate CENPA deposition at centromeres. We then review our knowledge of CENP-A ectopic distribution, highlighting experimental strategies that have enabled key discoveries. Finally, we discuss the implications of deregulated CENP-A in cancer.