One of agriculture’s major challenges is the low efficiency of phosphate (Pi) use, which leads to increased costs, harmful environmental impacts, and the depletion of phosphorus (P) resources. The TaPHT1;6 gene, which encodes a high-affinity Pi transporter (PHT), plays a crucial role in Pi absorption and transport. In this study, the promoter and coding regions of three TaPHT1;6 gene copies on chromosomes 5A, 5B, and 5D were individually amplified and sequenced from 167 common wheat (Triticum aestivum L.) cultivars. Sequence analysis revealed 16 allelic variation sites within the promoters of TaPHT1;6-5B among these cultivars, forming three distinct haplotypes: Hap1, Hap2, and Hap3. Field trials were conducted over two years to compare wheat genotypes with these haplotypes, focusing on assessing plant dry weight, grain yield, P content, Pi fertilizer absorption efficiency, and Pi fertilizer utilization efficiency. Results indicated that Hap3 represented the favored Pi-efficient haplotype. Dual-luciferase reporter assay demonstrated that the Hap3 promoter, carrying the identified allelic variation sites, exhibited higher gene-driven capability, leading to increased expression levels of the TaPHT1;6-5B gene. We developed a distributed cleaved amplified polymorphic site marker (dCAPS-571) to distinguish Hap3 from the other two haplotypes based on these allelic variation sites, presenting an opportunity for breeding Pi-efficient wheat cultivars. This study successfully identified polymorphic sites on TaPHT1;6-5B associated with Pi efficiency and developed a functional molecular marker to facilitate future breeding endeavors.