The Greek lentil landrace ‘Eglouvis’ is cultivated continuously at the Lefkada island for more than 400 years. It has great taste, high nutritional value and high market price. In the present study, we used morphological and molecular markers to estimate genetic diversity within the landrace. Morphological analysis was based on characteristics of the seed. Molecular analysis was performed using simple sequence repeat (SSR) molecular markers in a high-resolution melting (HRM) approach. ‘Samos’ and ‘Demetra’, two of the most widely cultivated commercial lentil varieties in Greece, were used for comparisons. Morphological analysis was performed with 584 seeds randomly selected from a lot. Analysis of seed dimensions and colour distributed the samples in different categories and highlighted the phenotypic variability in ‘Eglouvis’ lentil seeds. Genetic variability was estimated from 91 individual DNA samples with 11 SSR markers using HRM analysis. Genotyping was based upon the shape of the melting curves and the difference plots; all polymerase chain reaction products were also run on agarose gels. Genetic distances of individuals and principal coordinates analysis suggested that ‘Eglouvis’ landrace has a unique genetic background that significantly differs from ‘Samos’ and ‘Demetra’ and no overlapping could be detected. Genetic variability within the ‘Eglouvis’ landrace can be considered in targeted breeding programs as a significant phytogenetic resource of lentils in Greece.