Vascular plants appeared on land about 430 million years ago; one of the pivotal steps for the evolution of vascular plants was the acquisition of the mechanisms for making secondary walls-reinforced xylem conducting tissues. Dissecting the mechanisms controlling secondary wall biosynthesis may not only provide molecular tools for genetic modification of secondary wall-enriched biomass but also have implications in better understanding the evolution of vascular plants. In my dissertation, I used Selaginella moellendorffii, a model seedless vascular plant, and Physcomitrella patens, a model non-vascular plant, to investigate genes involved in secondary wall biosynthesis. It was found that S. moellendorffii xylan, one of the major secondary wall components, is substituted with methylated glucuronic acid, and xylans from both S. moellendorffii and P. patens are acetylated. Because gymnosperm xylans are non-acetylated, the finding that xylans from S. moellendorffii and P. patens are acetylated implies that the ability to acetylate xylans was acquired when land plants evolved, but this ability may be lost in the lineage of gymnosperms. There exist one DUF579 gene in P. patens and two DUF579 genes in S. moellendorffii, but only S. moellendorffii gene was found to encode a methyltransferase catalyzing xylan methylation, indicating that vascular plants recruited ancestral DUF579 genes as methyltransferases for xylan methylation. Complementation analysis of GT43 genes from P. patens and S. moellendorffii has revealed that the two GT43 genes in S. moellendorffii, SmGT43A and SmGT43B, are functional orthologs of Arabidopsis IRX9 and IRX14, respectively. This finding indicates that the involvement of two functionally non-redundant groups of GT43 proteins in xylan biosynthesis is evolutionarily conserved in both seedless vascular plants and seed plants. To investigate the evolutionary conservation of roles of secondary wall-associated NAC (SWN) transcription factors in secondary wall biosynthesis, functions of SWN genes from S. moellendorffii and P. patens were investigated through complementation and overexpression analyses. SmSWN and PpSWN genes were found to be functional orthologs of Arabidopsis NAC transcription factors and capable of activating the secondary wall biosynthetic pathway, indicating that SWN-mediated transcriptional regulation of secondary wall formation evolved as early as vascular land plants appeared.