The problem of exacerbating high levels of salinity should be considered greatest challenges facing Egyptian agriculture, especially in light of the limited water allocated for irrigation. Based on this, the Egyptian state has taken effective strategic steps through specialized institutes and research centers to improve the degree of tolerance of field crops to salt stress, especially the barley crop. The aim of the present work has concentrated on unraveling the genetic and biochemical factors and genetic markers that might be responsible or linked to salinity tolerance in barley. Some agro-morphological and biochemical attributes were the most studied parameters under both conditions. Profile of ISSR primers was used for comparing the ten barley genotypes (5 parents besides the best 5 crosses) confirming salinity tolerance in all studied traits under both experiments. The final results revealed the importance of the three first parents and the high five promising crosses were recorded high rank of salinity tolerance in this regard. Molecular markers have succeeded in reaching molecular genetic differences using ISSR primers which can be a taxonomic basis for determining tolerant barley genotypes to salinity and the other sensitive accessions. Where, the polymorphism % closed to 100% within using the ISSR primers; SR-03, SR-12 and SR-21 besides, generating 30 unique bands through all ISSR primers. Thus, the greatest benefit of this study may have been achieved, which is the strategy for improving barley crop to confront the danger of salt stress, which has become a major environmental problem, especially in the northern Delta regions. Further, its direct obstruction of agricultural development paths in Egypt.