CH257 is a threepistil material bred after three backcrosses between ‘threegrain wheat’ and common wheat ‘Shiyou 20’, which can greatly increase the number of grains per ear. In order to analyze the inheritance of three pistil trait in wheat, and dig out its control genes and develop related molecular markers, this study used CH257 and ‘China Spring’ to formulate a combination, and obtained its F₁, F₂ and BC₁F₁ populations for genetic analysis; using homozygous normal and homozygous threepistil F₂ lines, bulked segregant analysis (BSA) was used to construct singlepistil and threepistil mixed pools for 90K gene chip scanning, and molecular markers were developed in the corresponding segments based on the chip results. The results show that the development of the three pistils in CH257 is controlled by a pair of dominant single gene PisCH257. We located PisCH257 on the 2DL chromosome, and develop 5 markers in the corresponding segment of 2DL. The sequence of CH257linked codominant SSR markers is 2DL07, 2DL17, 2DL22, PisCH257, 2DL25 and 2DL38. the linkage markers 2DL22 and 2DL25 are on both sides of PisCH257 and their genetic distances are 1.1 cM and 2.5 cM, respectively. This study provides a basis for cloning the gene that control the three pistil trait of wheat.