The genetic diversity of Mesoamerican populations of the biofuel plant Jatropha curcas, using the fatty acids of the seeds as chemical markers was studied. The oil content of the whole seed in 135 accessions from 38 sites varied between 8.020% and 54.28%. The prevalent fatty acids were oleic acid (18:1) and linoleic acid (18:2), and the proportion of unsaturated fatty acids varied between 74.5% and 83.7%. A study with cloned plants grown in common garden showed that both the content of oil as well as the proportion of fatty acids are highly inheritable, therefore these chemical markers are valid for estimating the genetic diversity of the species. An analysis of principal components showed that the fatty acids that contribute more to the variance are stearic, oleic, linoleic, methylpalmitic, gadoleic and ricinoleic. The populations were classified in ten groups when the data were analyzed for fatty acids by analysis of clusters, showing the elevated genetic variation in natural populations of this native species of Mesoamerica. A discriminant analysis separated the populations in accordance with their geographic origin, which was verified with a Mantel test. Using the Monmonier’s algorithm two genetic barriers between the populations were identified. The results are discussed in light of their microevolutionary significance.