The goal of this research is to address the need for an alternative supply of jet fuel using native plants that do not compete with food crops. Pennycress (Thlaspi arvense), a member of the Brassicaceae, has been identified as a promising bioenergy crop: the chemical composition of its oil is ideally suited as a renewable source of biodiesel and jet fuel. This plant has tremendous potential for large‐scale oil production, has a short maturity time and is not a food crop. Pennycress can be commercially propagated as a winter annual, and has therefore the potential to be grown off‐season, complementing, rather than competing, the production of commodity crops, and serving as a winter cover crop. However, for this plant to become an economically viable source of specialty fuels, molecular and genetic resources need to be developed to facilitate and enhance oil production by breeding and/or genetic manipulation. This study aims to find potential biochemical step(s) that limit(s) oil synthesis, which will serve as targets for future crop improvement.To advance towards this goal, we determined the intracellular metabolite levels (metabolomics) in pennycress embryos at different stages of development. For this purpose, we have developed novel and highly sensitive methodologies using state‐of‐the‐art liquid chromatography tandem mass spectrometry (LC‐MS/MS). This metabolomics study highlighted the metabolites and pathways that were active in pennycress embryos and important for oil production. The contribution of each pathway to fatty acid synthesis in terms of carbon, reductant and energy provision is being assessed by measuring the carbon flow through the metabolic network. We are hence performing a 13C‐Metabolic Flux Analysis (fluxomics) in developing pennycress embryos to build a map of carbon flow through central metabolism. This study describes the combination of innovative tools that will pave the way for controlling seed composition in promising alternative crops.Support or Funding InformationJ.C.C was supported in part by the Agriculture and Food Research Initiative competitive grant # 2016‐67013‐24605 of the USDA National Institute of Food and Agriculture. This study is supported by the USDA‐U.S. Department of Energy Plant Feedstocks in Genomics for Bioenergy under Award Number DE‐SC0016490.