Plant R2R3-MYB transcription factors play an important role in plant secondary cell wall formation, by activating or repressing their target genes in a coordinated manner within a complex regulatory network. They belong to a large gene family - 180 genes in poplar -, with some members already identified as master switches in the regulation network (Zhang et al, 2014). Here, we combined genetic engineering and chromatin immuno-precipitation technique (ChIP-SEQ) in order to elucidate the function of 2 R2R3-MYB transcription factors in poplar, MYB090 and MYB156. MYB090 potentially regulates a high number of target genes in planta through a highly conserved motif, similar to GAMYB. These target genes are involved in the biosynthetic pathways of lignin, cellulose and xylans, which are the major components of secondary cell walls. Overexpression of MYB090 in poplar reduced stem growth and its total lignin content; lignification was severely impacted in wood parenchyma rays. By contrast, overexpression of MYB156 in poplar showed no growth defect, at least for some transgenic lines, while the total lignin content was decreased of 15% in the stem. These plants contained hypolignified fibers while the lignification of vessels seemed unaffected. These different results led to the conclusion that both MYB factors are transcriptional repressors of lignification, but act at different levels of the regulatory network. This work opens new avenues on the building of transcriptional regulatory networks involved in secondary cell wall formation. It also generated new poplar transgenic lines with interesting traits for bioethanol production. This work was carried out in the frame of AMC2B Région Centre Project and TreeForJoules Plant KBBE Project. WL received a PhD fellowship from Région Centre and INRA-EFPA.