AbstractThe pretreatment of biomass and the subsequent enzymatic hydrolysis to sugars play an important role in the production of biofuels from lignocellulosic biomass. However, the influence of pretreatment and hydrolysis yields on the production pathway performance of biofuels is rarely researched from the beginning. Moreover, a clear trade‐off between economic efficiency and environmental impact exists. Production pathways can be evaluated with reaction network flux analysis (RNFA) ( Voll A and Marquardt W, Reaction network flux analysis: Optimization‐based evaluation of reaction pathways for biorenewables processing. AIChE J 58(6):1788–1801 (2012)). Utilizing RNFA, this study explores the influence of biomass pretreatment, focusing on changes in biomass composition, fractionation efficiency, and sugar yield after hydrolysis on the production performance of biofuels for several pretreatment concepts and several wood sources. The results show that, for ethanol and ethyl levulinate production, specific fuel costs and carbon loss correlate reciprocally with the yields of pretreatment and hydrolysis. For a constant biofuel output, the main cost driver is the feed stream of biomass, which decreases with an improved overall sugar yield after pretreatment. Furthermore, above a threshold value, specific fuel costs increase strongly with carbon loss. As a result, a minimal yield of 40% carbohydrates from wood seems to be the limit of viable production in the processes that were considered. We therefore developed a facile strategy to assess the performance of pretreatment and hydrolysis in biomass processing © 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.