Background <p>Living xylem fibres play important physiological and mechanical functions in trees. Despite a significant role of secondary cell wall chemical composition in performing these functions, little information is available on the developmental changes in the cell wall of living fibres. In the present study, the distribution pattern of pectins, hemicelluloses, and lignin in the cell walls during the differentiation and maturation of living fibres in the secondary xylem of <i>Leucaena leucocephala</i> was examined by light and electron microscopy.</p> Results <p>The expansion of primary walls during the early stage of fibre development was characterised by a change in the organisation of pectic polysaccharides in the middle lamellae region. The intercellular regions became filled with pectic polysaccharides following initiation of secondary wall deposition. Subsequently, lignification started at cell corners with the deposition of guaiacyl units that co-polymerise with syringyl moieties in the final stages of fibre development. The transmission electron microscopic analysis confirmed the disorganisation of pectic polysaccharides in the middle lamellae region during cell expansion and their inhomogeneous distribution in the cell corners following secondary wall deposition. Immunofluorescence microscopy revealed that β(1–4)-galactan are mainly incorporated in the middle lamellae region that undergoes disorganisation and reorganisation during and after cell expansion. Immunogold labelling experiments using JIM5, JIM7 and CCRCM1 antibodies revealed dynamic changes in the distribution pattern of homogalacturonan with different degrees of methylation and fucosylated xyloglucan during cell wall loosening and secondary wall maturation stages of xylem fibres. In mature fibres, LM10 labelling indicated that the less substituted xylans are distributed throughout the secondary wall, while labelling of highly substituted xylans with LM11 appeared more intense at the corner regions of the secondary wall compared to other regions. The KMnO₄ staining revealed the relatively higher lignin distribution in xylem fibres in compound middle lamellae and S₃ wall layers. The transition zone between S₁ and S₂ layers showed relatively high lignin distribution compared to the rest of the S₂ wall layer. The ultrastructural studies demonstrated that the inhomogeneous distribution of lignin corresponds with that of pectins at the cell corners of fibres. The cell wall delignification resulted in a significant reduction of lignin at cell corners, compound middle lamellae and secondary wall layers of fibres.</p> Conclusion <p>This study revealed the dynamic developmental changes in the structure of the cell wall during secondary wall development by assembly of cell wall polysaccharides and lignin during differentiation of living fibres in <i>L. leucocephala</i>. These insights are very important to understand the development and functional dynamics of wood fibres.</p>