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In the conversion of woody biomass feedstocks into liquid fuel ethanol, the pretreatment process is the most critical and costly step. Variations in biomass composition based on genetic differences or environmental effects have a significant impact on the degree of accessibility accomplished by pretreatment and subsequent sugar release by enzymatic hydrolysis. To evaluate this, biomass from 10 genetically diverse, genotypes of shrub willow (Salix spp.) was pretreated with a hot-water process at two levels of severity, hydrolyzed using a combination of two commercial enzyme cocktails, and the release of hexose and pentose monomers was quantified by high-performance liquid chromatography. Among the genotypes selected for analysis, cellulose content ranged from 39 to 45% (w/w) and lignin content ranged from 20 to 23% (w/w) at harvest. Differences in the effectiveness of the pretreatment process were observed among the various willow genotypes. Correlations were identified between total sugar release and % cellulose and % lignin content. There was a significant effect of pretreatment severity on polysaccharide accessibility, but the response to pretreatments was different among the genotypes. At the high severity pretreatment 'SV1' was the least recalcitrant with sugar release representing as much as 60% of total biomass. These results suggest that structural, as well as chemical characteristics of the biomass may influence pretreatment and hydrolytic efficiency.