Background: Breeding for salinity tolerance in open-pollinated crops depends on broad genetic variation in order to select for vigorous plants under salinity stress. Objective: To compare the yield of some local alfalfa cultivars under saline and non-saline soils and to develop molecular genetic markers associated with salt tolerance using RAPD and ISSR analysis, thereby providing a basis for alfalfa salt-tolerance breeding and research. Methods: A two years field trial was carried out during 2013/14 and 2014/15 in Galbana Village at Sahl El-Tina site., North Sinai Governorate with 13 cuts after establishing period. Three local parental cultivars, nine selected populations for salt stress under different stress levels (three initial EC's: 8.73, 10.63 and 12.43 dSm^-1), a new tolerant pop, and three exotic varieties were included in the experiment under initial EC's (10.83). Results: Under Egyptian conditions, local cultivars: Siwa and Ismaelia were qualified as more adapted to salinity stress conditions than the exotic cultivars. New tolerant pop. ranked as the top yielding across all tested parental populations followed by Siwa and Ismaelia tolerant selected populations. New valley population had the lowest fresh, dry and protein yield overall studied populations. The tolerant selected populations were out yielded than their parental cultivars. A total of 16 alfalfa populations were analyzed at the molecular level to develop molecular markers associated with salt tolerance using seven RAPD and five ISSR primers. All primers succeeded to generate reproducible polymorphic DNA products. RAPD primers produced eight positive specific markers and six negative specific markers for salt tolerance in alfalfa population Sinai-1, while ISSR primers produced 12 positive specific markers and six negative specific markers for salt tolerance in alfalfa. The dendrograms were generated using pooled RAPD, ISSR, and the combined analysis of their data divided the alfalfa populations into two main clusters. The first cluster consisted of population Sinai 1 alone, while the second one contains all other evaluated populations. Conclusion: The new tolerant pop. could be considered as a new promising population and more adapted to salt stress.