RNF14 is a human RING-between-RING (RBR) E3 ubiquitin ligase that was found to be involved in ribosomal quality control (RQC) pathways. Recently, our lab discovered that RNF14 forms K6 and K48 linked ubiquitin, along with branched K6/K48 ubiquitin chains associated with the proteasomal degradation of eukaryotic release factor 1 (eRF1). This branching activity is proposed to be facilitated by RNF14’s intrinsically disordered C-terminal tail. To validate this, we compared RNF14 against its yeast orthologue Itt1. Itt1 is an active RBR E3 ubiquitin ligase lacking the disordered C-terminal tail region. Here, we show that unlike RNF14, Itt1 predominantly generates K6 ubiquitin chains in vitro. Further biophysical binding studies confirm the differing chain-type specificities of RNF14 and Itt1. These findings can be utilised to provide insights on RNF14’s K6 di-ubiquitin forming activity, a crucial step preceding the formation of K6/K48 branched tri-ubiquitin. By generating tail chimeras of RNF14 and Itt1, where we replace the C-terminal tail of one enzyme with that of the other, we show that the C-terminal tails define chain-type specificity and chain-branching activity. The generation of tail chimeras further enabled us to switch the activity and chain-type specificity of RNF14 with that of Itt1 and vice versa. Collectively, this study allows for a more comprehensive understanding of the mechanistic details behind RNF14’s K6/K48 branched ubiquitin chain forming activity, paving the way for downstream studies on the biological significance of these chain types in RQC.