Uncertainty analysis is used to understand the relative impact of measured variables on the accuracy of calculated values. In this study, we propagated the uncertainty in feedstock composition and process variables to the uncertainty in yields for pretreatment, enzymatic hydrolysis, and fermentation in a lignocellulosic biochemical conversion process. Using the uncertainty of feedstock composition and process flows obtained from experimental data as inputs, the values of uncertainty for the yields of relevant reactions were calculated. For pretreatment, the results indicate a 6-7% relative uncertainty in xylose yields and a 9-10% relative uncertainty in the yields of minor sugars. The primary contributors of uncertainty in pretreatment are the fraction insoluble solids (FIS), feed composition, and the feed and slurry flow rates. For enzymatic hydrolysis, the relative uncertainty is approximately 24% in the glucose yield. The uncertainty in FIS measurement contributes 99% of the total uncertainty. For fermentation, a relative uncertainty of 13% is observed for the ethanol yield with FIS contributing 99% of the total uncertainty. The results indicate that the uncertainty in the FIS measurement should be reduced by an order of magnitude before other uncertainties contribute significantly to uncertainties in yield in the enzymatic hydrolysis and fermentation. It is expected that these results will have a significant impact on the uncertainty in ethanol production cost estimates.