Mycobacteria comprises a large group of organisms including the pathogenic species Mycobacterium tuberculosis, the causative agent of tuberculosis. A fast- growing saprophytic member of this genus, however, Mycobacterium smegmatis, is oftentimes used as a model organism for the pathogenic species. With a unique cell envelope architecture and unconventional polar growth, spatial coordination of cell envelope biosynthesis is vital for proper assembly of this complex structure. Here, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain (IMD), a spatially distinct region of the plasma membrane with diverse functions. Besides harboring processes important for the cell envelope biosynthesis, previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we the confirmed the IMD-association of this biosynthesis in M. smegmatis implying a previously unappreciated role of the IMD linked to the central metabolism of mycobacteria. Finally, we show evidence that the same IMD exists inM. tuberculosis in addition to the classical plasma membrane. Similar to M. smegmatis, enzyme assays, western blotting, and proteomic analysis all indicated enrichment of lipid metabolism and other reactions in the IMD of M. tuberculosis. Taken together, these data indicate that functional compartmentalization of membrane is a common feature found in both M. tuberculosis and M. smegmatis. These observations support that the IMD is a conserved domain within bacteria involved in a variety of cellular processes.