The genome organizations of eight phylogenetically distinct species from five mammalian orders were compared in order to address fundamental questions relating to mammalian chromosomal evolution. Rates of chromosome evolution within mammalian orders were found to increase since the Cretaceous-Tertiary boundary. Nearly 20% of chromosome breakpoint regions were reused during mammalian evolution; these reuse sites are also enriched for centromeres. Analysis of gene content in and around evolutionary breakpoint regions revealed increased gene density relative to the genome-wide average. We found that segmental duplications populate the majority of primate-specific breakpoints and often flank inverted chromosome segments, implicating their role in chromosomal rearrangement.