Gentiana lutea L. (yellow gentian, Gentianaceae) is a protected orophyte of central and southern Europe, mainly threatened by the uncontrolled collection of its rhizome, used in traditional medicine and for liquor production. The species is self-compatible, but outcrossing mediated by pollinators is needed to obtain a viable progeny. In this study, we considered five natural populations belonging to the four subspecies of G. lutea. We performed controlled pollinations in the field (self- versus cross-pollination) followed by seed germination tests in laboratory conditions, adding a solution of gibberellic acid, in order to evaluate the seed performance. A cumulative index of inbreeding depression was calculated considering maternal reproductive output as well as seed performance traits. Seed weight and seed germination performance was similar between seeds resulting from naturally pollinated and pollen-augmented flowers and higher compared to selfed flowers, highlighting a disadvantage of selfing and the importance of cross-pollen transfer in natural conditions. However, in the small and isolated population of G. lutea subsp. symphyandra on Mt Grande we found a general reduction in seed germination rate, likely due to increased selfing or mating among close relatives as a consequence of a severe bottleneck. We discuss our results with regards to implications for conservation practices.