Faba bean (Vicia faba L.) is one of the most important legumes in the world. Little is known about the genetic resources of faba bean in Southern Tunisia. In the present study, genetic diversity within Tunisian faba bean germplasms was investigated using 16 simple sequence repeat markers. In total, 50 alleles were detected. The number of alleles per marker ranged from 2 to 6, with an average of 3. Genetic diversity and polymorphism information content values averaged, respectively, 0.43 (range 0.34–0.51) and 0.36 (range 0.28–0.43). The mean heterozygosity value was 0.27. A model-based structure analysis based on neighbour-joining tree and factorial correspondence analysis revealed the presence of two subpopulations, consistent with the clustering based on genetic distance (GD). The overall F
is value was 0.36, indicating the importance of selfing in these populations. Analysis of molecular variance revealed that the within-population genetic variance component was much higher than the between-population or between-subpopulation variance component. The genetic relationships based on Nei's GD revealed that AGD (Aguadulce) and SAG (Super Aguadulce) and TF1 and TF2 (Tafartassa-Gafsa) were the most closely related populations. Assessment of genetic variation within faba bean populations will be informative for the conservation of germplasms and the implementation of effective breeding programmes in Tunisia.