ABSTRACT Soil salinization is a severe abiotic stress that affects plant growth and development, leading to accelerated maturity and decreased production and quality. In the present study, we evaluated 170 Oenothera L. germplasms for saline‐alkali tolerance by subjecting them to 150 mmol·L −1 saline‐alkali stress (pH = 9.0). Germination parameters were analyzed, and comprehensive evaluation methods were used to assess their tolerance at the germination stage. Among the 170 germplasms, we identified 12 saline–alkali‐tolerant and 34 saline–alkali‐sensitive germplasms. Subsequently, 23 germplasms were further evaluated for saline‐alkali tolerance at the seedling stage. While seedling growth was inhibited across all specimens, five saline‐alkali‐tolerant germplasms were ultimately selected through comprehensive evaluation methods. We further examined photosynthetic pigment levels, membrane permeability, antioxidant oxidases, osmotic regulators, and mineral content to determine the plants' response. Germplasms with a higher tolerance to saline‐alkali stress can adapt by (i) maintaining or increasing chlorophyll content and delaying leaf senescence, (ii) using osmotic adjustment by maintaining stable osmotic potential, (iii) maintaining relatively stable electrical conductivity, (iv) enhancing antioxidant systems and maintaining a relatively low level of malondialdehyde, and (v) utilizing ion adjustment by promoting higher accumulation of Ca 2+ , Mg 2+ , and Mn 2+ in the roots and ensuring higher ratios of K + /Na + , Ca 2+ /Na + , and Mg 2+ /Na + in the leaves. These findings could serve as a valuable reference for future investigations of genetic mechanisms underlying saline‐alkali tolerance in Oenothera L. species and provide a foundation for the conservation of germplasm resources, genetic improvement, and application of Oenothera L. species in saline‐alkali environments.