RESEARCH SUMMARY Centromeric satellites and their binding partners are engaged in an ongoing evolutionary arms race such that each side is continuously evolving to win the race. As a result, centromeres are one of the most divergent genomic elements in populations, which ultimately stop exchanging genetic material and become two lineages. Highly intractable nature of repetitive centromeric satellite harboring loci has been an impediment to investigating the role of centromere evolution. The proposed research will test the role of centromere evolution in lineage separation by utilizing 1) a state-of-the-art genomics-based approach to study satellite evolution 2) geographically separated and partially reproductively isolated house mouse lineages, which serve as an ideal system to study lineage separation 3) a candidate based evolutionarily guided approach to delineate genetically complex process of the lineage separation. Interestingly, binding of centromeric proteins at centromeric satellites varies between these lineages despite the short evolutionary time after their split. This suggests that rapid divergence of centromeres in these two evolving species have acquired functional differences, which can be measured. This work proposes that rapidly evolving subspecies-specific variants of centromeric satellites and centromeric proteins result in functional centromere variation between house mouse lineages. This hypothesis predicts that a cross between these lineages generates chromosome segregation asymmetries in the hybrid meiosis due to “centromere incompatibilities”. Taking advantage of the measurable functional divergence, this proposal aims to identify lineage specific centromere features that contribute to the hybrid failure. Our multidisciplinary evolutionary guided approach combines genomics, cell biology, and genetics to study incompatibilities between centromeres of diverging lineages in the hybrid. The proposed research will not only help in understanding the role of centromere evolution in lineage separation but will also provide insights into the mechanisms of centromere evolution and inheritance. It will also enhance our understanding of widespread chromosomal disorders resulting from impaired centromere function. Centromere metabolism in cancerous cells involves expansion of satellite arrays and over-expression of centromeric proteins and mimics centromere evolution in nature. Findings from our research will thus provide insights into the process of centromere microevolution in cancers.