Pre-harvest sprouting (PHS) causes significant losses of both yield and quality, representing one of the major issues in cereal production worldwide. However, the molecular mechanism underlying PHS remains largely unknown, particularly in wheat. Here, we repetitively found a key QTL locus on Chr2A for PHS resistance through GWAS analysis using a population of 192 cultivars. Within this locus, a transcription factor TaMYB7 was suggested to be the candidate factor. Two haplotypes of TaMYB7 separate the 192 cultivars and Chinese mini core collection (MCC) with significant difference in germination rate of kernels within spike and mature seeds. A 221-bp CACTA type transposon insertion in the TaMYB7 promoter confers higher transcriptional level of TaMyb7 and more resistance to PHS among wheat varieties. This Triticum specific TE likely acts as an enhancer, regulating the transcriptional level of TaMYB7 associated with local chromatin landscapes alteration. Overexpression of TaMyb7 increased the resistance of PHS in transgenic wheat, further confirming the role of TaMYB7 in inhibition PHS. Moreover, through transcriptome, chromatin accessibility, binding motif analysis and reporter assays, we identified that TaABI4, transcription factor positively regulates ABA signaling, directly activates the expression of TaMYB7. TaMYB7 directly activates the expression of TaABI5 and inhibited the expression of TaAMY1 (α-amylase). In cultivars with different haplotypes of TaMYB7, we detected altered α-amylase activity and ABA signaling transduction in mature seeds. Thus, our study indicates that TaMYB7 plays important role in mediating wheat PHS via regulation of α-amylase activity and ABA signaling.