Most studies of exodermal structure and function have involved species with a uniseriate exodermis. To extend this work, the development and apoplastic permeability of Iris germanica roots with a multiseriate exodermis (MEX) were investigated. The effects of different growth conditions on MEX maturation were also tested. In addition, the exodermises of eight Iris species were observed to determine if their mature anatomy correlated with habitat.Plants were grown in soil, hydroponics (with and without a humid air gap) or aeroponics. Roots were sectioned and stained with various dyes to detect MEX development from the root apical meristem, Casparian bands, suberin lamellae and tertiary wall thickenings. Apoplastic permeability was tested using dye (berberine) and ionic (ferric) tracers.The root apical meristem was open and MEX development non-uniform. In soil-grown roots, the exodermis started maturing (i.e. Casparian bands and suberin lamellae were deposited) 10 mm from the tip, and two layers had matured by 70 mm. In both hydro- and aeroponically grown roots, exodermal maturation was delayed. However, in areas of roots exposed to an air gap in the hydroponic system, MEX maturation was accelerated. In contrast, maturation of the endodermis was not influenced by the growth conditions. The mature MEX had an atypical Casparian band that was continuous around the root circumference. The MEX prevented the influx and efflux of berberine, but had variable resistance to ferric ions due to their toxic effects. Iris species living in well-drained soils developed a MEX, but species in water-saturated substrates had a uniseriate exodermis and aerenchyma.MEX maturation was influenced by the roots' growth medium. The MEX matures very close to the root tip in soil, but much further from the tip in hydro- and aeroponic culture. The air gap accelerated maturation of the second exodermal layer. In Iris, the type of exodermis was correlated with natural habitat suggesting that a MEX may be advantageous for drought tolerance.