Drought priming, a potential strategy to bolster wheat yield amidst recurring droughts. Identifying the responsive cultivars and deciphering the underlying mechanisms are vital to better activate the instinct of plants coping with drought stress. Here, responses of 157 wheat cultivars to drought priming were phenotyped using a high-throughput phenotyping (HTP) platform across two wheat-growing seasons. A drought priming index (DPI) was devised to assess the drought priming sensitivity for each wheat cultivar. Based on the DPI comprehensive score (DPICS) derived from 13 identified sensitive traits by principal component analysis, the significant DPICS variations led to the classification of the cultivars into two distinct groups. Drought priming sensitive group contains 58 cultivars, demonstrated higher DPI values for traits such as yield components, harvest index, post-anthesis assimilation, photochemical efficiency, canopy coverage, and NDVI, but lower DPI values for traits like pre-anthesis dry matter remobilization, non-photochemical quenching, plant senescence reflectance index, and canopy temperature. Genome-wide association study (GWAS) based on DPI identified 499 significant markers related to drought priming using Wheat660 SNP arrays. Notably, one marker situated on chromosome 5B consistently appeared in both years. This marker resides within a 261.2 Kb genomic block containing seven genes, including the candidate gene TraesCS5B03G1259700, which exhibited distinct transcriptional memory related to drought priming. Our results suggest integrating HTP and GWAS has great potential for deciphering the genetic basis of acquired drought tolerance induced by priming and may facilitate breeding smarter wheat varieties responding to recurred drought evens.