Environmental stress, especially saline soils and saline water, are one of the most important limiting factors for agricultural crops in particular all around the world. Thus, yield enhancement in agricultural crops such as barley under saline conditions is a major goal of plant breeding. Leaf Sample from five barley genotypes and their F-1 offspring were collected at 30 days old seedlings growth under three treatments (control, 7000 and 14000 ppm) of NaCl to develop initial material for salinity tolerance breeding program using biochemical and molecular tools. These genotypes differ genetically in their salt tolerance potentiality and classified to salinity stress tolerant (Arar, Giza 123 and Giza 124), moderate (Mari) and susceptible (Beecher). Based on SDS-PAGE of water soluble protein for all genotypes under study, newly synthesized protein bands of salt treated parents observed at molecular weight (102, 96, 67 and 23) KDa and (28 and 87 KDa) for treated parents and hybrids, respectively. Also Native PAGE was carried out in this experiment to study six isozymes (EST, SKD, FDH, GDH. MDH and PER) patterns. In general theses isozymes patterns were reliable system for discriminating between tolerant and sensitive salinity genotypes under salt stress. Using RAPD-PCR with 5 primer arbitrary oligonucleotide (P18, P86, P24, P92 and P93), the results showed that all barley genotypes are not always identical in their DNA ability to be amplified and the total of amplified bands is 352 PCR bands. On the contrary, primers P18, P86 and P24 were able to generate positive marker, P92 was able to generate negative marker and 93 was able to generate positive and negative marker for salt tolerance. The phylogentic tree succeeded in clustering together the three tolerant parents and moderate parent while sensitive parents in another cluster. These results indicated that protein, isozyme and RAPD analysis are useful molecular tools to indicate genetic polymorphism between the barley genotypes under salt stress. [Metwali E. M. R. Molecular studies on some barley genotypes in relation to salt stress tolerance] Life Science Journal, 2012; 9(1):111-123] (ISSN: 1097-8135). http://www.lifesciencesite.com.