Quercus suber L. has been investigated with special attention to the stem bark and its cork formation layer, but excluding the roots. Roots are the location of infection by pathogens such as Phytophthora cinnamomi responsible for the tree's sudden death. It is widely accepted that suberin establishes boundaries within tissues, serves as a barrier against free water and ion passage, and works as a shield against pathogen attacks. We followed the suberization of young secondary roots of cork oak. The first suberin deposition detectable by transmission electron microscopy (TEM) and neutral red (NR) was in the endoderm Casparian strips. Casparian strips are not detected by Sudan red 7B and Fluorol yellow (FY) that specifically stain lamellae suberin. Reaction to Sudan was verified in the endodermis and later on in phellem cells that resulted from the phellogen. Under TEM, the Sudan and FY-stained cells showed clear suberin lamellae while the newer formed phellem cells displayed a distinct NR signal compared to the outermost phellem cells. We concluded that suberin chemical components are arranged differently in the cell wall according to the physiological role or maturation stage of a given tissue.