MAIN CONCLUSION: Secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Gene duplication is an important mechanism for functional divergence of genes. Secondary cell wall-associated cellulose synthase genes (CesA4, CesA7 and CesA8) are duplicated in Populus plants due to a recent whole genome duplication event. Here, we demonstrate that duplicate CesA genes show tissue-dependent expression divergence in Populus plants. Real-time PCR analysis of Populus CesA genes suggested that Pt × tCesA8-B was more highly expressed than Pt × tCesA8-A in phloem and secondary xylem tissue of mature stem. Histochemical and histological analyses of transformants expressing a GFP-GUS fusion gene driven by Populus CesA promoters revealed that the duplicate CesA genes showed different expression patterns in phloem fibers, secondary xylem, root cap and leaf trichomes. We predicted putative cis-regulatory motifs that regulate expression of secondary cell wall-associated CesA genes, and identified 19 motifs that are highly conserved in the CesA gene family of eudicotyledonous plants. Furthermore, a transient transactivation assay identified candidate transcription factors that affect levels and patterns of expression of Populus CesA genes. The present study reveals that secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules.