Lignin, one of the main cell wall components in plant xylem, is the most abundant natural aromatic polymer in the world, and it is expected to convert lignin into value-added materials by thermochemical processes such as hydrothermal treatment. For the efficient conversion, quantitative and/or qualitative modification of lignin by genetic manipulation is the promising strategies. Especially, the overexpression of frulate 5-hydroxylase (F5H) gene, involved in lignin biosynthesis, can increase the ratio of syringyl (S) to guaiacyl (G) units (S/G ratio). However, the change in the detailed lignin structures such as chemical linkages are not yet fully clarified. In this study, the transgenic poplar (Populus tremula × Populus alba) with various S/G ratios were prepared by the overexpression of F5H, and their lignin structure was analyzed. As a result, the transgenic lines with a variety of high S/G ratios from 2.2 to 11.7 were successfully prepared without any growth penalty and reduction of lignin content. Interestingly, the increase in β-O-4 linkage was limited and β-1 and β-β linkages elevated with the increase in S/G ratio. Furthermore, the hydrothermal treatment (230°C/10 min) was conducted to transgenic poplars. The solubilization yields were 45.4% and 51.1% for the wild type and S-rich poplar (Line 23), respectively. There was a positive correlation between the solubilization yields and S/G ratio, indicating that the changes of lignin unit structure caused these differences.