Unambiguous subunit assignment in a multicomponent complex is critical for thorough understanding of the machinery and its functionality. The eukaryotic group II chaperonin TRiC/CCT folds approximately 10% of cytosolic proteins and is important for the maintenance of cellular homeostasis. TRiC consists of two rings and each ring has eight homologous but distinct subunits. Unambiguous subunit identification of a macromolecular machine such as TRiC through intermediate or low-resolution cryo-EM map remains challenging. Here we present a yeast internal-subunit eGFP labeling strategy termed YISEL, which can quickly introduce an eGFP tag in the internal position of a target subunit by homologous recombination, and the tag labeled protein can be expressed in endogenous level. Through this method, the labeling efficiency and tag-occupancy is ensured, and the inserted tag is usually less mobile compared to that fused to the terminus. It can also be used to bio-engineer other tag in the internal position of a protein in yeast. By applying our YISEL strategy and combined with cryo-EM 3D reconstruction, we unambiguously identified all the subunits in the cryo-EM map of TRiC, demonstrating the potential for broad application of this strategy in accurate and efficient subunit identification in other challenging complexes.