Lignin is a major chemical compound of wood and one of the most abundant organic biopolymers on earth. It accumulates in the secondary cell wall of xylem cells and is a major target for tree breeders because of its foreseen role in the emerging bioeconomy. In this study, we paved the way toward an accelerated domestication of a widely grown tree species, Eucalyptus urophylla, by molecular breeding. To this end, we first described the pattern of nucleotide variation occurring at seven structural and regulatory genes of the lignin biosynthesis pathway and found high levels of average nucleotide and haplotype diversity per gene (?? =?0.0065 and Hd?=?0.853). Then, taking advantage of a pre-existing factorial mating design, a candidate-gene-based quantitative trait locus (QTL) detection strategy was used to compare the variation of lignin quality (syringyl by guaiacyl ratio (S/G)) with the nucleotidic variability in these seven genes in 304 genotypes belonging to 33 connected full-sib families. Two genes, encoding cinnamoyl-CoA reductase (CCR) and a Rho-like GTPase (ROP1), were shown to be linked to the variation of S/G through different single and multi-locus single-nucleotide polymorphism (SNP)- and haplotype-based association methods. Providing that relevant candidate genes are selected and their patterns of nucleotide diversity is accurately described, we showed that quantitative trait nucleotides (QTNs) can be detected taking advantage of pre-existing field experiments and trait measurements gathered in the framework of a forest tree breeding program.