Mentha longifolia L. is one of the most valuable perennial, rhizomatous, aromatic and herbaceous plants belonging to the Lamiaceae family. In this study, the effects of deficit irrigation stress on physiological traits and yield of 20 different wild mint populations were evaluated by a split-plot experiment based on a randomized complete block design with three replications. Soil moisture control was performed using irrigation treatments based on 100% (control) and 60% of field capacity. In the current study, physiological traits including chlorophyll a, b and total, carotenoids, proline, total carbohydrate, relative water content, ion leakage, stomatal conductivity, sodium and potassium elements, dry matter yield and essential oil yield were investigated. There were significant differences (P<0.01) between populations in two levels of water deficit treatment. The most tolerant populations had the highest essential oil yield (4.69%), relative water content (69.91%), total chlorophyll (0.32 mg/g) and proline (2.80 µmol/g) under water deficit stress. Results of correlation between dry matter yield and all traits except carotenoid content were significant. Finally, five populations of Hormozgan, Khuzestan, South Khorasan, Kerman-1 and Lorestan respectively were identified as the tolerable populations and introduced for future breeding programs.