Fig (Ficus carica L.) tree is cultivated worldwide and is highly appreciated for its fruit, which is consumed fresh or dried, having high nutritional and pharmaceutical value and for these reasons there is an increasing interest for its cultivation. In the present study, an ex situ collection of 60 fig accessions (41 indigenous Greek and 19 from other Mediterranean countries) was established and its diversity was analyzed using eight simple sequence repeat (SSR) loci. Greek fig genotypes showed relatively low allelic variation (the average number of SSR alleles per locus was 3.75), an excess of heterozygosity (mean He = 0.489 and Ho = 0.557), and extensive outbreeding (mean F index − 0.151). Cluster analysis showed that the established fig population exhibited weak genetic structure, with most of the genetic variation (89%) being present within individual members of the clusters. Both cluster and principal coordinate analysis confirmed that there is little correlation between genetic makeup and geographical origin of the fig accessions. Polymorphism information content with an average of 0.421 was reasonably informative. An identification key scheme for fig cultivars that will be useful in cultivar discrimination and intellectual property protection was developed. This work will contribute to a sustainable fig production regionally and worldwide, through the establishment and conservation of a reference fig collection, providing germplasm for future breeding efforts.