Despite the growing resources and tools for high-throughput characterization and analysis of genomic information, the discovery of the genetic elements that regulate complex traits remains a challenge. Systems genetics is an emerging field that aims to understand the flow of biological information that underlies complex traits from genotype to phenotype. In this study, we used a systems genetics approach to identify and evaluate regulators of the lignin biosynthesis pathway in Populus deltoides by combining genome, transcriptome and phenotypic data from a population of unrelated individuals of P. deltoides. The discovery of lignin regulators began with the quantitative genetic analysis of the xylem transcriptome and resulted in the detection of 6,706 and 4,628 significant local- and distant-eQTL associations, respectively. Among the locally-regulated genes, we identified the R2-R3-MYB transcription factor MYB125 as a putative trans-regulator of the majority of genes in the lignin biosynthesis pathway. The gene expression of MYB125 in a diverse population positively correlated with lignin content. Furthermore, overexpression of MYB125 in transgenic poplar resulted in increased lignin content, as well as altered gene expression of structural genes in the lignin biosynthesis pathway. Altogether, our findings indicate that MYB125 is involved in the control of a transcriptional co-expression network of lignin biosynthesis genes during secondary cell wall formation in P. deltoides.