Faba bean (Vicia faba L.) is an important grain legume with high protein content (approximately 25–30%) and high nutritional value. It is broadly cultivated in temperate areas both for human consumption and as animal feed. According to FAOSTAT (2020), the total cultivated area of faba bean reached approximately 2.5 million ha, yielding more than 4.5 million tons. The characterization of the genetic diversity in faba bean is an important parameter for genetic and biodiversity studies, germplasm characterization, and for introducing genetic variability in plant breeding. The present study aims to assess the genetic diversity among 53 Greek, varied faba bean populations provided by the Hellenic Agricultural Organization “DEMETER” seed bank. To determine the genetic diversity of the studied populations, six SCoT DNA markers were used. A total of 114 loci were obtained with 37.95% being polymorphic and 62.05% monomorphic within or between populations. SCoT markers are a useful tool for the detection of genetic diversity among faba bean populations and encourage targeted crossing strategies. The present study is the first step towards the development of an efficient breeding program.