Iris germanica roots develop a multiseriate exodermis (MEX) in which all mature cells contain suberin lamellae. The location and lipophilic nature of the lamellae contribute to their function in restricting radial water and solute transport. The objective of the current work was to identify and quantify aliphatic suberin monomers, both soluble and insoluble, at specific stages of MEX development and under differing growth conditions, to better understand aliphatic suberin biosynthesis. Roots were grown submerged in hydroponic culture, wherein the maturation of up to three exodermal layers occurred over 21 days. In contrast, when roots were exposed to a humid air gap, MEX maturation was accelerated, occurring within 14 days. The soluble suberin fraction included fatty acids, alkanes, fatty alcohols, and ferulic acid, while the suberin poly(aliphatic) domain (SPAD) included fatty acids, α,ω-dioic acids, ω-OH fatty acids, and ferulic acid. In submerged roots, SPAD deposition increased with each layer, although the composition remained relatively constant, while the composition of soluble components shifted toward increasing alkanes in the innermost layers. Air gap exposure resulted in two significant shifts in suberin composition: nearly double the amount of SPAD monomers across all layers, and almost three times the alkane accumulation in the first layer. The localized and abundant deposition of C18:1 α,ω-dioic and ω-OH fatty acids, along with high accumulation of intercalated alkanes in the first mature exodermal layer of air gap-exposed roots indicate its importance for water retention under drought compared with underlying layers and with entire layers developing under water.