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Genus Medicago belonged to family Fabaceae is one of the most important forage legumes. Annual species of this genus are indigenous to Mediterranean region and used for prevention of soil erosion, green manure and forage. In this research, genetic variability and classification of 14 annual medicago truncatula genotypes using ISSR markers was done. 9 out of 15 ISSR primers which possessed suitable polymorphism and amplification were used for fingerprinting of studied genotypes. Totally, 71 bands were amplified via ISSR primers which 11 bands were monomorphic and 60 bands were polymorphic across genotypes. Based on Jaccard similarity coefficient, minimum similarity (0.25) was seen between TN8.3 (Tunisia) and TN6.18 (Tunisia) and maximum similarity (0.82) was seen between TN8.3 (Tunisia) and SA28064 (Cyprus). Population structure analysis using STRUCTURE software subdivided them into 9 subpopulation. In this study, maximum admixture was occurred in TN1.21 (Tunisia), A10 (Australia), F83005-5 (France), SA22322 (Syria), A20 (Australia), and DZA315-16 (Algeria) genotypes. Results revealed that annual self-pollinated M. truncatula had noticeable genetic variation which Is accurately detectable using ISSR molecular markers

Keywords: Annual medicago, ISSR marker, population structure, Genetic variability

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