The Renewable Fuels Standard (RFS) and Energy Independence and Security Act of 2007 (EISA) mandated that 36 billion gallons of biofuels should be blended into transportation fuel by 2022. Implementing this will help reduce greenhouse gas emissions, reduce petroleum imports and encourage the development and expansion of US renewable fuels sector within rural America. Of the 36 billion gallons of biofuels, 16 billion gallons is expected to be from lignocellulosic biomass such as trees and grasses. The Black Hills of South Dakota is rich in ponderosa pine. This feedstock for bioethanol production, which is widely available due to recent pine beetle infestation, will not only add to the RFS requirement, it will also have a positive impact on rural economies in South Dakota. From the wood chips of pine, after acid pretreatment and enzymatic hydrolysis, the fermentable sugars obtained are relatively dilute in concentration (∼20-30 g/L). Hence, within a biorefinery, to increase the fermentation efficiency and decrease downstream processing cost of the biofuels, concentrating the sugars can be beneficial. In this study, Reverse Osmosis (RO) and Nanofiltration (NF) membranes were tested with complex lignocellulosic hydrolysate samples for their ability to concentrate sugars prior to fermentation. Fouling analysis and membrane characterization for both RO and NF membranes were performed by SEM, AFM, BET, contact angle and FTIR spectroscopy. Efficiency of membranes for their ability to separate fermentation inhibitors (e.g., organic and mineral acids, furans and phenolic compounds) from sugars, while simultaneously concentrating the sugars was studied to make the bio-ethanol production process cost and energy efficient. Three commercial nanofiltration membranes GE-R, TS40 and SR100 showed very promising results. GE-R concentrated sugars to more than 2.5 fold in the retentate, and simultaneously separated more than 50% of the inhibitory components into permeate. These results will increase the fermentation efficiency and reduce downstream purification costs of the produced fuel.