Wheat pre-harvest sprouting significantly reduces yield production and flour quality. It has been gradually aggravated in recent years， and destabilize the safety of wheat production in China， particularly in the Huanghuai region. Identification of pre-harvest sprouting resistant germplasm and functional molecular markers are of importance to accelerate pre-harvest sprouting resistant breeding. In this study， 77 pre-harvest sprouting resistant germplasms observed from field experiments and 128 advanced breeding lines from a rotational population introduced with six resistant germplasms were used. These genotypes were tested for pre-harvest sprouting resistance using the whole-split germination and seed germination methods， as well as genotyped by functional markers Vp1B3， Dorm-B1， and PM19. 49.35% （38 of 77） of germplasm resources showed intermediate resistance， and 57.90%（22） of them contained functional resistance alleles of Vp1Ba or（and） Dorm-B1b. The germplasm resources Xinong 172， Kalango， Huaimai 40 and Yunong 186 were detected carrying both two functional alleles. 36.72% （47） of the advanced breeding lines showed intermediate resistance， of which 87.23% contained resistance alleles and 17.02% （8） contained two functional alleles. Stacking resistance loci can increase the level of pre-harvest sprouting resistance. In the germplasm resources， the whole-split germination and seed germination decreased from 36.65% and 34.99% in non-resistant individuals， respectively， to 18.17 % and 23.87 % in individuals with two resistance loci. The same pattern has been observed in advanced lines. There was a significant difference in the number of resistant loci between advanced materials and others. Among them， 17.02% of the materials with medium resistance level contained two resistance loci， while only 4.94% of the other materials without medium resistance level contained two resistance loci. This study deployed molecular markers and phenotypic characterization techniques to identify pre-harvest sprouting resistance germplasm， followed by germplasm innovation using dwarf failing rotation selection， which provided a basis for future improvement of wheat spike germination resistance in the Huanghuai region in China.