Climate change poses a significant challenge to agriculture while barley is an essential and crucial crop worldwide. This study evaluated the drought stress tolerance of 25 barley genotypes. A field experiment was carried out to investigate agronomical traits, such as plant height at 110 days (PH110) and spike length (SL), in response to different surface irrigation treatments. Subsequently, 15 barley genotypes were chosen for the second experiment which aimed to examine the impact of physiological stress generated by polyethylene glycol-6000. Several biological metrics, including seedling vigor index (SVI), and drought tolerance index (DTI), were quantified. Ultimately, six SSR primers were used to analyze the genetic diversity between different barley genotypes. The findings demonstrated that the G1, G2, and G6 genotypes were tolerant but G5, G9, and G14 were susceptible. The primers Bmag0603, EBmac0849, and Bmag770 were polymorphic. This study provides valuable initial insights into the drought resistance of various barley genotypes, highlighting the genetic diversity and potential for breeding drought-tolerant varieties. We suggest expanding the sample size and incorporating a broader range of environmental conditions in future studies to validate these findings. Additionally, the identified genetic markers could be further explored and utilized in breeding programs.