Background: Jatropha curcas is an oilseed plant with great potential for biodiesel production. In agricultural industry, the seed quality is still estimated by manual inspection, using destructive, time-consuming and subjective tests that depend on the seed analyst experience. Recent advances in machine vision combined with artificial intelligence algorithms can provide spatial and spectral information for characterization of biological images, reducing subjectivity and optimizing the analysis process.Results: We present a new method for automatic characterization of jatropha seed quality, based on multispectral imaging (MSI) combined with X-ray imaging. We propose an approach along with X-ray images in order to investigate internal problems such as damages in the embryonic axis and endosperm, considering the fact that seed surface profiles can be negatively affected, but without reaching important internal regions of the seeds. Our studies included the application of a normalized canonical discriminant analyses (nCDA) algorithm as a supervised transformation building method to classify spatial and spectral patters according to the classes of seed quality. Spectral reflectance signatures in a range of 780 to 970 nm and the X-ray images can efficiently predict quality traits such as normal seedlings, abnormal seedlings and dead seeds.Conclusions: MSI and X-ray images have a strong relationship with physiological performance of Jatropha curcas L. These techniques can be alternative methods for rapid, efficient, sustainable and non-destructive characterization of jatropha seed quality in the future, overcoming the intrinsic subjectivity of the conventional seed quality analysis.