The multimerization of ubiquitins at different positions of lysine residues to form heterotypic polyubiquitin chains is a post-translational modification that is essential for the precise regulation of protein functions and degradative fates in living cells. The understanding of structure-activity relationships underlying their diverse properties has been seriously impeded by difficulties in the preparation of a series of folded heterotypic chains appropriately functionalized with different chemical tags for the systematic evaluation of their multifaceted functions. Here, we report a chemical diversification of enzymatically assembled polyubiquitin chains that enables the facile preparation of folded heterotypic chains with different functionalities. By introducing an acyl hydrazide at the C terminus of the proximal ubiquitin, polyubiquitin chains were readily diversified from the same starting materials with a variety of molecules, ranging from small molecules to biopolymers, under nondenaturing conditions. This chemical diversification allowed the systematic study of the functional differences of K63/K48 heterotypic chains based on the position of the branch point during enzymatic deubiquitination and proteasomal proteolysis, thus demonstrating critical roles of the branch position in both the positive and negative control of ubiquitin-mediated reactions. The chemical diversification of the heterotypic chains provides a robust chemical platform to reframe the understanding of how the ubiquitin codes are regulated from the viewpoint of the branch structure for the precise control of cell functions, which has not been deciphered solely on the basis of the linkage types.