By converting edible oils into biodiesel, food resources are species actually being converted into automotive fuels. A non-edible oilseeds such as the perennials jatropha, might contribute to greening of wastelands without compromising food and fodder security. Jatropha has never been domesticated and its yield is difficult to predict with accuracy. There is limited information available agronomy of Jatropha and also a lack of benchmark descriptors and information on genetic variability, effects of environment and genotype x environment interaction. Improved varieties with desirable traits for specific growing conditions are not available, which makes growing jatropha a risky business. An in vitro germplasm collection of 1300 accessions from 13 countries was used to study genetic diversity and the relationship between and within Jatropha species. Ecotilling was applied to 12 different genes of interest related to stress tolerance, toxin and oil metabolism. 50 ISSR and 36 AFLP primers were used to assess the genetic diversity of Jatropha curcas and related species. Due to the reported low variation between Jatropha accessions and large size of the collection, 8 x 8 pooling strategy was chosen to estimate the level of variations among selected genes. 4 AFLP and 5 ISSR primers yielded highly reproducible patterns. Ecotilling is a low cost, high-throughput reverse genetic method for haplotyping and SNPs discovery. The level of differentiation observed was based on the geographic distribution pattern (it was higher in the centre of origin). The data showed clear variations not only among individuals but also between different regions.