The root system is a major organ for water and nutrient uptake. Exploration and utilization of the genes that regulate root development are important for enhancing wheat production. bHLH (basic Helix-Loop-Helix), a transcription factor family widely present in plants, plays an essential role in regulating the growth and development of plants. In this study, the TabHLH123-6A gene was isolated based on sequence homology, with an open reading frame of 1386 bp that encodes a protein comprises of 461 amino acids containing a conserved HLH domain. Based on the prediction of interacting proteins, TabHLH123-6A might be able to interact with HLH family members and zinc finger proteins. qRT-PCR analysis revealed the transcript of TabHLH123-6A in various tissues of wheat at different stages, with higher expression level in roots and root bases than that of other tissues. The promoter sequence analysis detected a number of cis-acting elements, including hormone response elements such as auxin, abscisic acid and methyl jasmonate. The expression of TabHLH123-6A was inducible under the treatments of auxin, abscisic acid or methyl jasmonate, but significantly repressed by PEG treatment. A nucleotide variation (C/T) at 25 bp of TabHLH123-6A gene (designated as TabHLH12325-C/T) was found using a panel of wheat accessions. A functional marker dCAPS-KpnI of TabHLH123-6A was developed to target this polymorphic site. Association analysis showed this polymorphism being significantly correlated with the ratio of deep root in wheat. The ratio of deep root with TabHLH12325-C allele is higher than that of TabHLH12325-T, and the frequency of TabHLH12325-T has been positively selected in the breeding process. Collectively, this study provided the theoretical basis and a locus for the improvement of wheat root system architecture.