In flooded soils, an efficient internal aeration system is essential for root growth and plant survival. Roots of many wetland species form barriers to restrict radial O2 loss (ROL) to the rhizosphere. The formation of such barriers greatly enhances longitudinal O2 diffusion from basal parts towards the root tip, and the barrier also impedes the entry of phytotoxic compounds produced in flooded soils into the root. Nevertheless, ROL from roots is an important source of O2 for rhizosphere oxygenation and the oxidation of toxic compounds. In this paper, we review the methodological aspects for the most widely used techniques for the qualitative visualization and quantitative determination of ROL from roots. Detailed methodological approaches, practical set-ups and examples of ROL from roots with or without barriers to ROL are included. This paper provides practical knowledge relevant to several disciplines, including plant–soil interactions, biogeochemistry and eco-physiological aspects of roots and soil biota.