Triticum aestivum L. with three pistils (TP) is characterized by three normal stamens and three fertile pistils, which can produce 2~3 seeds per floret and significantly increase the number of grains per spike. Therefore, developing specific molecular markers for early and accurate identification of the three-pistil trait is of great significance for marker-assisted selection breeding. Based on the unique structural variation in the 589.7~590.1 Mb region of chromosome 2D in three-pistil wheat, we extracted the deleted region and breakpoint sequences, and designed primers targeting the conserved sequences of normal single pistil wheat genome (SP1, SP2) and primers spanning the variant breakpoints (SV1, SV2), respectively. After PCR validation of their specificity in the Fielder×TP F₃ population, four specific markers were obtained. Subsequently, using 23 segregating populations of three-pistil wheat and their parents (a total of 255 experimental materials) as subjects, we established an early molecular identification system and verified the effectiveness of marker-assisted selection through PCR amplification and agarose gel electrophoresis, combined with field phenotypic investigation. The results showed that primer pairs SP1 and SP2 could specifically identify normal single pistil wheat genotype, whereas SV1 and SV2 were effective for distinguishing three-pistil wheat materials. Mutual validation between the two sets of markers confirmed the genotyping results and effectively reduced detection errors. The consistency between molecular detection results of the 255 materials using the developed markers and field phenotypic identification reached 96.08%. The molecular markers developed from chromosome-specific structural variations demonstrated high specificity and stability, enabling accurate early screening at the seedling stage. These markers provide valuable technical support for marker-assisted breeding and germplasm exploitation of three-pistil wheat.