Mitoribosomes are specialized for the synthesis of hydrophobic membrane proteins encoded by mitochondrial DNA, all essential for oxidative phosphorylation. Despite their linkage to human mitochondrial diseases and the recent cryo-EM reconstruction of yeast and mammalian mitoribosomes, how ribosomes are assembled remains obscure. Here, we dissected the yeast mitoribosome large subunit (mt-LSU) assembly process by systematic genomic deletion of 44 mt-LSU proteins (MRPs). Analysis of the strain collection unveiled 37 proteins essential for functional mt-LSU assembly, three of which are critical for mt-LSU 21S rRNA stability. Hierarchical cluster analysis of mt-LSU subassemblies accumulated in mutant strains revealed cooperative assembly of protein sets forming structural clusters and preassembled modules. The analysis also indicated crucial roles for mitochondrion-specific membrane-binding MRPs in anchoring newly transcribed 21S rRNA to the inner membrane, where assembly proceeds. Our results define the yeast mt-LSU assembly landscape in vivo and provide a foundation for studies of mitoribosome assembly across evolution. As this assembly map is generated using mutant strains, which has the caveat that in some instances it may deviate from the native pathway, to further complete the study of mitoribosome assembly in yeast, we are planning experiments to identify native 54S assembly intermediates in WT cells. For that purpose, we will use stable isotope labeling with amino acids in cell culture (SILAC) followed by quantitative mass spectrometry, an approach that should help to refine the assembly map obtained from analyzing the mutant strains. A major difference between MRPs and their homologous bacterial ribosomal proteins is that most of the MRPs contain an N- or C-terminal mitochondrial specific extension whose role is yet to be uncovered. The collection of MRP mutant strains provide a useful tool to study the function of these extensions. Our preliminary data showed that most of the extensions are essential for cell respiration and mitochondrial protein translation, suggesting the importance of these extensions for mitoribosome assembly. Additional studies on these extensions will define their function and role in mitoribosome assembly.