In order to study the effect of drought stress on seedling morpho-physiological traits of alfalfa (Medicago sativa), the seeds of ten populations were collected in their natural habitats from different parts of the Fars province, Iran in 2013. A factorial experiment was conducted using 10 alfalfa populations and four water stress treatments of 100, 75, 50, and 25% Field Capacity (FC) based on a completely randomized design with three replications in the glasshouse of agriculture and natural resources research center of Fars province, Iran. Data collected for shoot and root fresh and dry weight, root and stem length, Root/Stem length ratio (RS) and free proline content. Data were subjected to analysis of variance, cluster analysis, principle component analysis. The main effect of population and water stress treatments were significant for all of traits (P<0.01) and population by water stress interaction effects were significant only for proline and root dry weight (P<0.01). The results showed high variation among populations under different water stress treatments. The means comparison among populations for all the water stress treatments showed that population of Kamal Abad Neiriz had high seedling growth coupled with long root length and was considered as drought tolerance to water stress that could be used to improve new varieties. The higher heritabilities were obtained for stem length (h2=0.52), root length (h2=0.48.8) and proline content (h2=0.47.7). According to cluster analysis, the four populations of Deh Kohneh, Dozkord, Kamal Abad, and Komhar that were placed into cluster 2 had higher mean values for many traits than other populations/clusters. This high variation could be used for breeding programs and making synthetic populations for improving alfalfa productivity. Manuscript profile