Abstract Trees can generate large mechanical stresses at the stem periphery to control the orientation of their axes. This key factor in the biomechanical design of trees, named “maturation stress”, occurs in wood fibres during cellular maturation when their secondary cell wall thickens. In this study, the spatial and temporal stiffening kinetics of the different cell wall layers were recorded during fibre maturation on a sample of poplar tension wood using atomic force microscopy. The thickening of the different layers was also recorded. The stiffening of the CML, S 1 and S 2 -layers was initially synchronous with the thickening of the S 2 layer and continued a little after the S 2 -layer reached its final thickness as the G-layer begins to develop. In contrast, the global stiffness of the G-layer, which initially increased with its thickening, was almost stable long before it reached its final maximum thickness. A limited radial gradient of stiffness was observed in the G-layer, but it decreased sharply on the lumen side, where the new sub-layers are deposited during cell wall thickening. Although very similar at the ultrastructural and biochemical levels, the stiffening kinetics of the poplar G-layer appears to be very different from that described in maturing bast fibres. Highlight New insights into the changes in mechanical properties within the cell wall of poplar tension wood fibres during maturation have been obtained using atomic force microscopy.