Hordeum marinum commonly known as sea barley is a salinity-tolerant species of grass. In the current study, 150 lines from ten populations of H. marinum ssp. marinum collected from five Tunisian bioclimatic sites were screened for polymorphism with 13 selected random amplified polymorphic DNA primers. Results exhibited a high level of polymorphism (160 polymorphic bands with an average of 12.46 per primer) and a high level of genetic diversity in all the studied populations (on average UHe = 0.247 and I = 0.358). High discrimination capacity was found for the 13 primers and a combination of three allowed assignation of a unique profile for each of the 150 lines. The partition of genetic diversity with Analysis of Molecular Variance suggested that the majority of genetic variation (67%) was within populations. The components between-populations within ecoregions and between-ecoregions explained 21 and 12%, respectively, of the total genetic variance. There was no significant association of population differentiation (ФPT) with geographical distance or altitudinal difference. Results also showed that the 150 lines grouped into three clusters with no respect to geographic origin. A sub-set of 13 lines was identified, which captured the maximum genetic diversity of the entire collection. The genetic variation found in this collection of H. marinum is deemed to be useful in formulating conservation strategies for this species.