The biofuel market is dominated by ethanol and biodiesel derived from cellulosic and lipid-based biomass crops. This is largely due to the relatively low costs and reliability of production. At present, production of non-food plant-derived oils for biofuel production in the U.S. is minimal. A research team from the Commonwealth Scientific and Industrial Research Organization (CSIRO), an independent Australian federal government research institution, has developed an efficient transgenic system to engineer oil production in tobacco leaves. This novel system is comprised of multiple transgenes that direct the endogenous metabolic flux of oil precursors towards triacylglycerol (TAG) production. Additional genes were incorporated to store and protect the accumulated oil in vegetative tissues. Preliminary greenhouse tests by the CSIRO research group indicated an oil content of > 30% by dry weight (DW) in tobacco leaf lamina. Here we evaluated two transgenic lines against a non-transgenic control in 2017 and 2018 in greenhouse and field production systems. The 2017 pilot study showed that the high leaf-oil tobacco line was viable and will grow in the field in Kentucky. Chemical analyses revealed significantly higher oil content compared to the non-transgenic control despite several logistical setbacks. These promising discoveries prompted the deployment of additional transgenic line assessments and further data validation in 2018. Line evaluations in 2018 revealed that the LEC2:WRI1:DGAT:OLE transgenic line had the highest leaf oil content (≥ 19.3% DW-1) compared to both the WRI1:DGAT:OLE transgenic line (≤ 5.6% DW-1) and non-transgenic control (≤ 2.1% DW-1). The results of this research will contribute to the successful development of transgenic tobacco lines engineered to accumulate high concentrations of TAG in the leaves.