In an effort of overcoming the limited availability of fossil energy resources the focus of the research and development in the area of biofuels has moved towards developing the second generation of fuels that should be produced via microbial fermentation. The idea is to use as a feedstock inexpensive and abundant waste materials such as lignocellulosic biomass. The second generation biofuels should satisfy several criteria such as lower emission, higher energy density and should be less corrosive to engines. However, currently used industrial workhorses such as E. coli and S. cerevisiae do not produce many of these molecules naturally. Furthermore, for majority of these molecules there are no known biochemical pathways. The need for discovery of novel biosynthetic pathways towards desired molecules sparked the development of computational tools that are capable of reconstructing feasible reaction steps between a given set of starting compounds (precursors) and a molecule of interest (a target compound). In this study, we performed the retrobiosynthesis analysis for all the candidates applying Biochemical Network Integrated Computational Explorer (BNICE.ch) and we reconstructed the metabolic network for 69 fuel compounds. We analyzed compounds (biological or chemical) and reactions one-step away from these target molecules and the appearance of the potential substrates in their reconstructed metabolic network. Based on the results of this analysis, coupled with other criteria such as the Gibes free energy of formation, combustion potential and expert opinion, we chose the 5 highest ranked fuel candidates for further pathway reconstruction studies. For these 5 fuel candidates we have created 1.8 million de novo pathways with different number of reaction steps for further evaluation. We have then embedded the reconstructed pathways in the genome-°©‐scale models of the two chassis organisms E. coli and P. putida and we performed the pathway evaluation with respect to their maximum theoretical yield using two methods (i) Flux Balance Analysis (FBA), and (ii) Thermodynamic-based Flux Analysis (TFA). All the pathways that did not satisfy the thermodynamic constraints were rejected. Our results indicated that in the evaluation and ranking of the pathways the thermodynamics is a necessary consideration. Specifically, we showed that the majority of the pathways that were determined as feasible based on FBA, they were not feasible based on TFA. Moreover, we also showed that some pathways that were determined as feasible in both organisms based on FBA, they were not feasible in both of them based on TFA. Also, the yield of the feasible pathways can differ depending on the organism. These results demonstrate how TFA can also guide the selection of a suitable chassis organism. For all discovered reactions in the feasible pathways we have also identified known enzymes from the KEGG database with the closest reaction similarity that could be engineered for the implementation of novel reactions. This study shows the full potential of computational tools for discovery and design of novel synthetic pathways and their relevance for future developments in the area of metabolic engineering and synthetic biology.