Jatropha curcas L. has been promoted as a “miracle” tree in many parts of the world, but recent studies have indicated very low levels of genetic diversity in various landraces. In this study, the genetic diversity of landrace collections of J. curcas was compared with the genetic diversity of the species from its native range, and the mating system was analyzed on the basis of microsatellite markers. The genetic diversity parameters were estimated, and analysis of molecular variance, principal coordinate analysis, and unrooted neighbor-joining tree were used to describe the relationship among populations. Results confirmed very low genetic diversity in African and Asian landraces. Mexican populations from the regions of Veracruz, Puebla, and Morelos were also found to have low levels of diversity (mostly monomorphic), while populations from Chiapas were polymorphic with an expected heterozygosity between 0.34 and 0.54. Bayesian analysis showed differentiation according to geographic locations, which was confirmed by principal coordinate analysis and neighbor-joining tree. Estimations of outcrossing rate of individual families from Chiapas showed that some mother trees were mainly outcrossing. Mating system could not be estimated in the landraces from Mali and populations from Veracruz, Puebla, and Morelos (Mexico), as these were highly monomorphic. The observed low level of genetic diversity in some of the populations and landraces suggests that breeding programs should test for genetic variation and heritability in relevant quantitative traits and estimate if sufficient gain can be expected from traditional testing and selection. Diversification of the local gene pools may be considered for breeding and selection.