In the context of chemical ecology, the analysis of the temporal production pattern of volatile organic compounds (VOCs) in root tissues and the emission rate measurement of root-emitted VOCs are of major importance for setting up experiments to study the implication of these compounds in biotic interactions. Such analyses, however, remain challenging because of the belowground location of plant root systems. In this context, this study describes the evolution of the root VOC production pattern of barley (Hordeum distichon L.) at five developmental stages from germination to the end of tillering and evaluates the emission of the identified VOCs in an artificial soil. VOCs produced by crushed root tissues and released by unexcavated root systems were analysed using dynamic sampling devices coupled to a gas chromatography-mass spectrometry methodology (synchronous SCAN/SIM). The results showed that, at each analysed developmental stage, crushed barley roots produced mainly four volatile aldehydes: hexanal; (E)-hex-2-enal; (E)-non-2-enal; and (E,Z)-nona-2,6-dienal. Higher total and individual VOC concentrations were measured in 3-day-old seminal roots compared with older phenological stages. For each developmental stage, the lipoxygenase (LOX) activity was greater for linoleic acid than α-linolenic acid and the greatest LOX activities using linoleic and α-linolenic acids as substrates were measured in 7- and 3-day-old roots, respectively. The analysis of VOCs released by barley roots into the soil showed that (E)-non-2-enal and (E,Z)-nona-2,6-dienal were the only VOCs emitted in quantifiable amounts by mechanically injured roots.