The characterization of C. sativus ecotypes is of great interest for preserving them from a possible genetic erosion due to the decrease of European cultivation surface. In this study, we evaluated four ecotypes from Italy (Sardinia and Abruzzo), Spain (Castilla-La Mancha), and Greece (Kozani) in order to detect the existence of variability and promote the biodiversity of this crop. Thirty-one traits related to saffron flowering, flower morphology, production of spice and daughter corms, vegetative development (leaf and corm traits), and spice quality, were evaluated. In addition, a genetic analysis through three PCR-based approaches, SSRs, RAPD, and SRAP was assessed. Results highlighted a phenotypic variation among ecotypes during two consecutive years. All the studied parameters were influenced by the ecotype except for the stamen length, color coordinates of tepals, leaf length, and leaf number per plant. Sardinia had a longer flowering interval, earlier flowering, and higher spice yield and quality than the other corm origins. The maximum values of morphological traits, such as stigma length, dry weight of stigmas, tepals, flowers and leaves, leaf area, and daughter corm weight were observed in the Abruzzo ecotype. Principal component analysis (PCA) showed a clear separation among ecotypes, in which Sardinia and Spain showed more similarities than Abruzzo and Kozani. Significant negative correlation was found between days to flower with stigma yield and quality. However, we could not find molecular markers discriminating among corm origins. In conclusion, this study suggests the importance of C. sativus ecotypes as precious source of biodiversity and bioactive compounds, and of their enhancement as fundamental prerequisite for a sustainable development strategy and as an agricultural diversification opportunity for growers.