Drought stress is one critical limitation on global wheat production. Early drought priming events could enhance plant tolerance to re-occurring drought stress. In a solution culture experiment, 110 cultivars from Jiangsu (the mid-downstream region of Yangtz River), Shandong, Henan and Hebei provinces (the Huang-Huai-Hai Plain) were introduced to a moderate osmo-priming and a full recovery, and then subjected to a severe osmotic stress. Responses of the morphological and physiological traits to osmo-priming and osmotic stress were analyzed, including leaf relative water content, leaf area, shoot fresh and dry weight, root fresh and dry weight, root morphological, contents of proline content, total soluble sugars and sucrose. Moreover, phenotyping instruments of MultisqeQ were used to monitor leaf temperature, chlorophyll fluorescence parameters of ФPSII, Fv/Fm, ФNPQ and ФNO. The effectiveness of osmo-priming in alleviating the re-occurring osmotic stress was evaluated using principal components analysis and cluster analysis. The mean values of all the morphological traits were higher in primed plants than non-primed plants under osmotic stress, while reversed for leaf temperature, ФNPQ and ФNO. Leaf temperature and ФNO of most primed plants were significantly lower, while leaf relative water content, leaf area, shoot fresh and dry weight, root fresh and dry weight, proline content, total soluble sugar content, sucrose content and ФPSII were significant higher than the non-primed plants in response to the osmotic stress. The priming coefficients (the ratio of the primed plants to the non-primed plants??) varied between varieties, especially for the priming coefficients of contents of soluble sugars and proline, and of ФPSII. Meanwhile, strong correlation existed among different drought priming indexes. It is concluded that the osmo-priming was effective in improving osmotic stress tolerance of a majority of the wheat varieties. The effectiveness of osmo-priming of the tested varieties was then classified.