AbstractStomata are important channels for the control of gas exchange between plants and the atmosphere. To examine the genetic architecture of wheat stomatal index, we performed a genome‐wide association study (GWAS) using a panel of 539 wheat accessions and 450 678 polymorphic single nucleotide polymorphisms (SNPs) that were detected using wheat‐specific 660K SNP array. A total of 130 SNPs were detected to be significantly associated with stomatal index in both leaf surfaces of wheat seedlings. These significant SNPs were distributed across 16 chromosomes and involved 2625 candidate genes which participate in stress response, metabolism and cell/organ development. Subsequent bulk segregant analysis (BSA), combined with GWAS identified one major haplotype on chromosome 2A, that is responsible for stomatal index on the abaxial leaf surface. Candidate gene association analysis revealed that genetic variation in the promoter region of the hexokinase gene TaHXK3‐2A was significantly associated with the stomatal index. Moreover, transgenic analysis confirmed that TaHXK3‐2A overexpression in wheat decreased the size of leaf pavement cells but increased stomatal density through the glucose metabolic pathway, resulting in drought sensitivity among TaHXK3‐2A transgenic lines due to an increased transpiration rate. Taken together, these results provide valuable insights into the genetic control of the stomatal index in wheat seedlings.