In this study, 66 Aegilops cylindrica genotypes collected from West (non-saline) and Northwest (saline) Iran were investigated in four experiments. In the first experiment, genetic variation was evaluated using morphological traits. Genetic variation of 44 genotypes using 35 microsatellite (SSR) primer pairs was assessed in the second experiment. Cluster analysis based on SSR markers divided studied genotypes into two groups which were consistent with their geographical origin of saline and non-saline . Salinity tolerance of 44 genotypes was evaluated under control and 400 mM NaCl conditions using physiological traits. Results showed a significant variation in genotypes for the traits except for proline content. Results of linear regression analysis revealed that Na + exclusion is one of the most important physiological attributes of Ae. cylindrica in salinity tolerance. Six important salinity tolerance genes, AecHKT1;5 , AecSOS1 , AecNHX1 , AecVP1 , DREB2 and P5CS were characterized for the sequences and the expression profiles in the fourth experiment. Expression patterns of these genes using quantitative real-time PCR revealed a higher activity of AecHKT1;5 and AecSOS1 genes in roots than shoots while AecNHX1 and AecVP1 genes expressed ubiquitously in the whole plant. Stepwise multiple regression analysis showed that AecHKT1;5 and plant Na + concentration were the strongest predictors of dry matter (DM), and collectively explained 51% of DM variation under 400 mM NaCl treatment.