The gene pool of wild barley species is relatively rich during natural selection and evolution in fertile crescent-shaped areas under conditions of abiotic stresses such as drought and high salinity. This study was conducted in three separate experiments with the ultimate goal of evaluating the salinity tolerance of the lines resulting from the backcrossing of the cultivated barley ( Hordeum vulgare . vulgare ) and the wild barley ( H. vulgare . spontaneum ) and identification of microsatellite-associated markers related with salinity tolerance. The first study evaluated diversity of 47 spontaneum genotypes along with Rihan 03 and 6 hybrid F1 (crossing with cultivated with wild barley). The results of the first experiment emphasized the importance of the species spontaneum as a resource of valuable breeding genes for salinity tolerate in barley. The second study evaluated 47 spontaneum genotypes and 6 cultivated barley for salinity tolerance at the vegetative stage in the greenhouse conditions (control and 300 mM) and in the reproductive stage under field conditions (control and 23 dS m –1 ). In greenhouse conditions, the dry weight of roots and shoot, plant height, membrane stability index, malondialdehyde, proline, sodium, potassium and calcium elements on the roots and shoots, potassium to sodium ratio and chlorophyll fluorescence were measured. The results showed that the genotypes of spantaneum (wild) barley had a higher salinity tolerance than cultivated barley and identified the top 7 salinity tolerant genotypes of wild barley. Evaluation of salinity tolerance in the field under normal conditions and salinity stress was performed using lattice 8 × 8 design with 2 replications in two years. The results of field experiments also showed the superiority of spontaneum barley over barley cultivars in terms of having the lowest yield reduction and sodium concentration and the highest ratio of potassium to sodium. Despite having the lowest yield among cultivars, the Mona cultivar (two-rowed barley) ranked second in yield reduction due to its early maturity and escaping from salinity stress. The average maturity date in the two-rowed barley was less than the six-row barley. Based on the results, four wild genotypes including genotype numbers 23, 27, 42 and 43 were recognized as the best salinity tolerant ones at both the vegetative and reproductive stages. On the other hand, six cultivars along with several wild genotypes were grouped as salinity-sensitive genotypes. In the third experiment, the BC2F2 population derived from cross between the wild barley and cultivated barley was used. DNA samples from the BC2F2 population (142 lines) were used to genotype microsatellite markers. The phenotypic data were obtained from the 142 BC2F2 lines along with parents using a lattice 12 × 12 design in two normal and salt-stressed environments under field conditions. Cluster analysis of BC2F2 lines in normal and saline environments was divided the genotypes studied into three distinct groups, with one group having the highest grain yield, biological yield, harvest index, 1000-grain weight, and potassium to sodium ratio under saline conditions as well as the lowest yield reduction due to salinity. Twenty-one SSR and EST-SSR marker in the population amplified 77 loci , which were used to genotype 142 BC2F2 lines, which result in 7 linkage groups. The markers covered 1321.5 CM from the entire genome, with an average distance of two markers covering about 17.2 map unit (CM). QTL locations and effects for various attributes were obtained by two methods of single marker analysis and location of composite composite distance spacing (ICIM). The results of single marker analysis in salinity stress conditions, found the length of the spike, the weight of the spike, the weight of 1000 grains, the number of graier spike and the days to heading, proline and potassium with R2 10. In the ICIM method, a total coverage of 31 and 28 QTLs with a LOD above 2.5 were detected under normal conditions and salinity stress for physiological traits including sodium, potassium, potassium to sodium ratio, proline, peroxidized hydrogen and malondialdehyde, respectively. A genomic region at the end of a 1H chromosome contained six traits that were effective in tolerating salinity, indicating the importance of this region of genome for the simultaneous manipulation of several traits related to salinity tolearnce. The markers found to be related to salinity tolerance in this study can be further verified for marker-assisted selection in improving salinity tolerance in barley. Keywords Backcrossing, Hordeium spantaneum , QTL mapping, Salinity tolerance, Wild barley