The Italian region of Molise is characterised by a remarkable variety of microenvironments, where some olive cultivars have settled in the course of many centuries. The study of the varieties cultivated in Molise, and of their relationships with varieties of close regions, is very important in order to clarify the entity of the native olive germplasm, well adapted to the local conditions, and to characterise the oil with regional origin. Eighteen cultivars from Molise have been examined using AFLP markers. They were compared with other cultivars, widely cultivated in Italy and France, in order to evaluate their similarities and to test some hypotheses on their origin and spreading among different areas of cultivation. Numerous varieties showed a high similarity and one case of synonymy (Frantoio-Paesana Bianca) was observed. The results provide useful information in order to distinguish the native germplasm and evaluate the consistency of the olive heritage of the region.