A large amount of lignocellulosic biomass such as leaf litter residues and bagasse are generated during the sugarcane harvest or after the sugar refining process, respectively. Therefore, lignocellulosic biomass from leaf and processing residues will likely become a valuable feedstock for future biofuel production. However, lignin is recognized as the major limitation to efficient conversion of lignocellulosic biomass to biofuel. Therefore, altering lignin composition or reducing lignin content via RNAi suppression of lignin biosynthetic genes is a promising strategy to increase the efficiency of biofuel production from lignocellulosic sugarcane residues. In the lignin pathway, 4-coumarate-CoA ligase (4CL) and caffeic acid 3-O-methyltransferase (COMT) are key enzymes that catalyze the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates or the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde, respectively. In this study, COMT and 4CL genes were isolated from the commercially important sugarcane cultivar CP 88-1762 by a combination of cDNA library screening and PCR based approaches. More than 100 transgenic lines harboring COMTi or 4CLi or both COMTi and 4CLi constructs were generated via biolistic gene transfer. Quantitative real-time PCR identified transgenic lines ranging from no suppression to almost complete suppression of the target genes. Accumulation of siRNAs was confirmed in 23 transgenic lines by Northern blot analysis of low molecular weight RNA. These transgenic lines were vegetatively propagated and are currently grown to maturity in a replicated and randomized design. The analysis of the Klason lignin content will be carried out in the Spring of 2011 and presented at the conference. These results demonstrate that RNAi is effective in suppression of individual or co-suppression of multiple endogenous genes of the complex sugarcane genome. Further these results allow a determination of the relative importance of the targeted alleles or gene families for lignin biosynthesis and biofuel applications in sugarcane.