Seed oil production in Jatropha seeds through different maturity stages have been investigated. In order to meet the high demand of oil (feedstock) for large scale biodiesel production, increasing oil content or output in Jatropha seeds is required. Jatropha fruits were harvested at four different maturity stages and the seeds were analysed for oil content. The seed oil was analysed for fatty acid profile. Results from four different geographical locations investigated namely; Mmadinare, Thamaga, Maun and Shashe, have shown a similar trend in lipid accumulation in Jatropha seeds as the fruits mature from green to brown dry. However, maximum oil content in seeds varies with geographical location. Accumulation of oil in Jatropha seeds during maturation follows a parabolic trend and reaches its peak when fruits are yellow. Oil yield in Jatropha seed kernels ranges from 38.7% to 45.8% for the four maturity stages investigated. Overall results have revealed that harvesting Jatropha fruits when they are yellow increases seed oil output by 6-9% when compared to harvesting the fruits when they are brown dry. There is a relationship between the trend in fatty acid composition in Jatropha seed oil and seed oil content trend during fruit maturation. Based on the trend of unsaturated fatty acids in Jatropha seed oil, particularly linoleic and oleic acids, it can be deduced that reduction of seed oil content from yellow brown to brown dry stage is a result of breakdown of some of the unsaturated fatty acids.