Due to climate change, drought has become a severe abiotic stress that affects the global production of all crops. Elucidation of the complex physiological mechanisms underlying drought tolerance in crops will support the cultivation of new drought-tolerant crop varieties. Here, two drought-tolerant lines, RIL70 and RIL73, and two drought-sensitive lines, RIL44 and RIL93, from recombinant inbred lines (RIL) generated from maize drought-tolerant line PH4CV and drought-sensitive line F9721, were selected for a comparative RNA-seq study. Through transcriptome analyses, we found that gene expression differences existed between drought-tolerant and -sensitive lines, but also differences between the drought-tolerant lines, RIL70 and RIL73. ZmbHLH124 in RIL73, named as ZmbHLH124^T-ORG which origins from PH4CV and encodes a bHLH type transcription factor, was specifically up-regulated during drought stress. In addition we identified a substitution in ZmbHLH124 that produced an early stop codon in sensitive lines (ZmbHLH124^S-ORG). Overexpression of ZmbHLH124^T-ORG, but not ZmbHLH124^S-ORG, in maize and rice enhanced plant drought tolerance and up-regulated the expression of drought-responsive genes. Moreover, we found that ZmbHLH124^T-ORG could directly bind the cis-acting elements in ZmDREB2A promoter to enhance its expression. Taken together, this work identified a valuable genetic locus and provided a new strategy for breeding drought-tolerant crops.