Thirty-nine genotypes of Hordeum spontaneum were selected from three geographically separated areas (southwestern, northern, and southeastern) of the Fertile Crescent. The lines were subject to AFLP (amplified fragment length polymorphism) analysis from which a similarity matrix was produced. A dendrogram of the data showed, with two exceptions, that the genotypes grouped together according to area of origin. This was confirmed by principal coordinate analysis in which the first principal coordinate separated the genotypes of the southwestern area from the other two areas, which in turn could be separated by the second principal coordinate. While genotypes from the same site of origin can exhibit very similar AFLP profiles, sharp genetic differences were detected between genotypes separated by relatively short distances. Thirty of the 39 genotypes were subjected to hydroculture salt tolerance tests. These were analysed for shoot Na+ content and carbon isotope composition (δ13C) after 4 weeks of treatment (100 mol∙m−3 NaCl). Shoot Na+ content and δ13C were highly correlated. Twelve AFLP markers were found to be associated with both shoot Na+ content and shoot δ13C and were also associated with site of origin ecogeographic data, particularly longitude. The most salt tolerant genotype came from Ilam in the southeastern area and the most salt sensitive genotype originated in the southwestern area. The 12 markers were partitioned into groups that showed significant associations within groups but no significant association between groups. In a multiple regression analysis, three AFLP markers, from separate groups, accounted for more than 60% of the variation for shoot Na+ content and δ13C. The results demonstrate the effectiveness of AFLP fingerprinting in genetic studies of complex traits at the wild species and (or) population level.