To assess the genetic diversity among Tunisian local barley, a set of 120 barley accessions representing five distinct geographical regions (North-West, Littoral, South, Jerba and Kerkennah Islands) was characterized with 20 simple sequence repeats (SSR) and 8 expressed sequence tag (EST)-SSR markers. The 28 loci revealed a total of 98 alleles, with an average of 3.76 alleles per locus (range 2–10). Gene diversity averaged 0.50 (range 0.09–0.84). Partitioning analysis of genetic diversity showed that about 95% of the total variation was within regions and no geographical differentiation could be found except for the North-West population. Similarly, neighbour joining clustering of the genotypes did not indicate any clear pattern of division among the barley accessions based on geographical origin. These results may reflect the impact of seed exchange between farmers which is likely to limit highlighting favourable alleles due to local adaptation.