Abstract Recent increases in energy demands as a consequence of population growth and industrialization, and pollution caused during the extraction and combustion of fossil fuel sources have driven the development of new energy sources that do not cause pollution and are inexpensive and renewable. Consequently, it is necessary to develop alternative ways of generating biofuels that put less pressure on agricultural lands and water supplies, and ensure ecosystems conservation. In order to achieve the proposed goals related to energetic coverage and independence, several approaches have been developed, including biodiesel production using vegetal oils as feedstock. The aim of the current research project was to apply a nonconventional bioprocess for in vitro biomass and oil production of Jatropha curcas, for assessing different J. curcas varieties, where seed tissue was isolated and used for callus induction. Once friable callus was obtained, cell suspension cultures were established. The cell viability, fatty acid content, and characteristics were used to select the most promising cell line according to its fatty acid profile and ability to grow and develop under in vitro conditions. Oil produced by cell suspension culture of the Jatropha varieties studied was extracted and characterized by GC/MS. Differences encountered among Jatropha varieties were related to their fatty acid profiles, oil content (% on dry basis), and cell viability measurements (%).