ABSTRACT Dating back to the 1960s, initial studies on the staphylococcal cell wall were driven by the need to clarify the mode of action of the first antibiotics and the resistance mechanisms developed by the bacteria. During the following decades, the elucidation of the biosynthetic path and primary composition of staphylococcal cell walls was propelled by advances in microbial cell biology, specifically, the introduction of high-resolution analytical techniques and molecular genetic approaches. The field of staphylococcal cell wall gradually gained its own significance as the complexity of its chemical structure and involvement in numerous cellular processes became evident, namely its versatile role in host interactions, coordination of cell division and environmental stress signaling. This chapter includes an updated description of the anatomy of staphylococcal cell walls, paying particular attention to information from the last decade, under four headings: high-resolution analysis of the Staphylococcus aureus peptidoglycan; variations in peptidoglycan composition; genetic determinants and enzymes in cell wall synthesis; and complex functions of cell walls. The latest contributions to a more precise picture of the staphylococcal cell envelope were possible due to recently developed state-of-the-art microscopy and spectroscopy techniques and to a wide combination of -omics approaches, that are allowing to obtain a more integrative view of this highly dynamic structure.