We give the first account concerning the ecology, population size, breeding system and genetic variability of the unique Italian population of Gentianella crispata, a rare orophilous species with a disjunct distribution including Balkan peninsula and southern Italy. The population is relatively small (ca. 4000 individuals) and has a fragmented structure within a wider area covered by the grass-dominated vegetation Seslerio nitidae—Brometum erecti. The evaluated morphological traits (i.e. plant size, flower number per plant and length of corolla tube) greatly varied among individuals. Examination of the floral structures revealed that the stigma becomes receptive before anther dehiscence. Observations on the topological relationships between stigma and anther suggested that in young flowers protogyny is paralleled by herkogamy (i.e. hyper-stigmatic condition). However, the anther dehiscence appeared to be accompanied by a reduction of the spatial separation between female and male organs. This suggested the occurrence of a mixed breeding system, as also sustained by ISSR analysis. The ITS-based phylogeny showed an interesting relationship with G. caucasea, conferring a peculiar evolutionary interest to the species and its westernmost, severely isolated Italian population. According to the IUCN Red List criteria, G. crispata in Italy meets the requirements for critically endangered species.