This study was designed to understand how drought stress impact on the physiological and molecular status of genetically diverse Lolium perenne L. (perennial ryegrass) populations. The sensitivities of the 36 perennial ryegrass populations were checked against PEG-6000-simulated drought stress by analysing physiological status. Afterward, drought-related gene expressions were evaluated under normal and stressed conditions. The drought stress significantly up-regulated the drought-related genes in P6, P10, P15 and P30, while no difference was observed in drought-sensitive ones. The results collectively showed that four populations (P6, P10, P15, P30) are tolerant to drought stress and activate drought-related genes. Genetic similarities of the populations were also monitored using microsatellite (SSR) markers. The ten SSR primers detected 38 alleles. Surprisingly, the drought-tolerant populations were classified into different groups by principal component (PCA) and cluster analysis, showing that they are genetically different. Moreover, four unique alleles were identified by two SSR primers (P-07, and P-08) in P6, P10 and P15. In addition, the drought-tolerant populations (P6, P10, P15, P30) can be used to develop new perennial ryegrass cultivars for areas having drought problems.