Plant biodiversity must be safeguarded because it constitutes a resource of genes that may be used, for instance, in breeding programs. Lentil (Lens culinaris Medik.) is one of the most ancient crops of the Mediterranean region. Extensive differentiation of L. culinaris over millennia has resulted in a myriad of different landraces. However, in more recent times many landraces have disappeared consequent to environmental and socioeconomic changes. To promote the survival of endangered lentil landraces, we have investigated the genetic relationship between two ancient landrace cultivated in Capracotta and Conca Casale (Molise, south-central Italy) and widely spread commercial varieties using an integrated approach consisting of studies at morphological, DNA and protein level. Seeds of these two landraces were collected from local farmers and conserved in the Molise germoplasm bank. The two local landraces were well differentiated from each other, and the Conca Casale landrace was separated from the commercial varieties at morphological, protein and DNA level. The Capracotta landrace, was well separated from the commercial varieties, except Castelluccio di Norcia, at DNA level showing a more complex and heterogeneous segregation at morphological and biochemical level. The correlation between morphological, DNA and protein data, illustrates that proteomics is a powerful tool with which to complement the analysis of biodiversity in ecotypes of a single plant species and to identify physiological and/or environmental markers.