The midbody is a transient structure that connects two daughter cells at the end of cytokinesis, with the principal function being to localize the site of abscission, which physically separates two daughter cells. Despite its importance, understanding of midbody assembly and its regulation is still limited. Here we describe how the structural composition of the midbody changes during progression throughout cytokinesis and explore the functional implications of these changes. Deriving from midzones, midbodies are organized by a set of microtubule interacting proteins that colocalize to a zone of microtubule overlap in the center. We found that these proteins split into three subgroups that relocalize to different parts of the midbody: the bulge, the dark zone, and the flanking zone. We characterized these relocalizations and defined domain requirements for three key proteins: MKLP1, KIF4, and PRC1. Two cortical proteins-anillin and RhoA-localized to presumptive abscission sites in mature midbodies, where they may regulate the endosomal sorting complex required for transport machinery. Finally, we characterized the role of Plk1, a key regulator of cytokinesis, in midbody assembly. Our findings represent the most detailed description of midbody assembly and maturation to date and may help elucidate how abscission sites are positioned and regulated.