Fig (Ficus carica L.) considers the original cultivated fruit trees and currently has become extinct. Such genetic resources should be identified, documented, and conserved. Morphology, pomology, and molecular markers are successful tools in assessing genetic diversity and classifying fig accessions. Twenty-one cultivated fig (F. carica L.) accessions were collected from Egypt and Libya. In Egypt, fig accessions are dispersed from Sinai in the east to El-Saloom in the west and from Alexandria in the north to Aswan in the south, whereas Libyan accessions were collected from Tubryq, Bengazi, and AlKufrah. Seventeen morphological, pomological, and fruit traits were used to characterize the fig accessions. Moreover, frozen young leaves were used to extract genomic DNA; 13 expressed sequence tag (EST) common fig primer pairs with 12 intersimple sequence repeat (ISSR)-anchored primers were used to detect the genetic diversity. Analysis of variance for fig accessions showed highly significant differences concerning morphological traits, i.e., the leaf length (centimeters) and width (centimeters) ranged from 5.4 and 6 cm to 23 and 23.5 cm, for Komesrey-El-Hammam, Abodey-Giza, and Black_Mission accessions, respectively. Also, fig accessions showed different shapes of leaf edge and fruits; they were categorized into four groups: straight, waved, zigzag, and serrated. The number of leaf lobes data ranged from one lobe for the ‘Green-yellow’, ‘Sultani Red Siwa’, and ‘Sultany Red Amria’ accessions to 10 lobes in the Aswany accession. The two-way hierarchical morphological cluster analysis distributed fig accessions into two main groups. The results detected high genetic diversity for the fig accessions that could be useful in the future breeding programs. Concerning molecular data, the EST markers showed highly polymorphism and informative (r = 0.61; 90.0%), with a total number of identified alleles of 78. We proved that a relatively greater number of alleles per locus characterizes the targeted loci among fig accessions, for which only one and two alleles per locus have been revealed, respectively, although ISSR showed a clear pattern and bands of the primers UBC807, UBC811, UBC812, UBC814, UBC815, UBC817, UBC818, and UBC823. In conclusion, a great range of variability was detected within the fig accessions. This diversification could enrich the genetic base of this genus, and more experiments are needed to reach its full potential.