Grain number is a critical factor influencing yield and reproductive coefficient in wheat hybrid breeding. Unlike common wheat, the three-pistil wheat, first described in 1983,

is an unusual germplasm characterized by the presence of three pistil per floret, which consequently formed three close-set grains in a back-to-back manner after pollination. Here, we generated a 14.63 gigabase chromosome-scale assembly of the tri-pistil wheat cultivar TP1 using high-coverage PacBio HiFi sequencing, and identified a 411.41 kb deletion and a 30.00 kb insertion ~21.73 kb upstream of the causal gene TaWUS-D1, which encodes a WUSCHEL-like homeobox transcription factor. These structural variations reduce DNA methylation levels in the promoter region and cause activation of TaWUS-D1 during spike development. Field assessments showed that the tri-grain trait increased grain number per spike by over 40%, with equivalent production yield. This study resolves a long-standing genetic mystery and provides a valuable target for wheat hybrid breeding programs.