We identified a wheat (Triticum aestivum L.) multi-pistil mutant from an F2 breeding population in 2012, named 12TP (three pistils in one floret). Genetic analysis showed that one dominant gene locus controlled the multi-pistil trait. Using homozygous normal and multi-pistil lines (near-isogenic lines; NILs) derived from the original mutant 12TP, a simple sequence repeat (SSR) marker assay located the 12TP locus on chromosome arm 2DL. Four SSR markers were linked to 12TP and their order was Xcfd233→Xcfd62-12TP→Xwmc41→Xcfd168 at 15.85, 10.47, 2.89, and 10.37 cM, respectively. The average genetic expressivity of the trait ‘three pistils in one floret’ was more than 98% in seven homozygous 12TP lines; however, the average genetic expressivity in heterozygous F1 plants was about 49%. Thus, the 12TP is a semi-dominant gene locus, which differ from all previously reported multi-pistil mutants. Mutant 12TP is a new useful germplasm for study of wheat floral development and for breeding of high yield wheat.