Wheat pre-harvest sprouting reduces yield and flour quality significantly. It has been gradually aggravated in the Huanghuai region in recent times, jeopardizing the safety of wheat production in China, particularly in the Huanghuai region. Screening for pre-harvest sprouting resistant germplasm and available functional molecular markers is key to accelerating pre-harvest sprouting resistant breeding and mitigating its damage. In this study, 77 pre-harvest sprouting resistant germplasm identified naturally over many years and 128 advanced lines created from a rotational population introduced with five resistant germplasms were used as materials for pre-harvest sprouting resistance identification and evaluation by the whole-split germination and seed germination methods, as well as for identifying the distributions of the functional markers of spike germination, Vp1B3, Dorm-B1, and PM19, in the germplasm and evaluating the breeding effect of these markers. The results showed that 49.35% (38 out of 77) of germplasm resources reached the level of intermediate resistance, and 57.90% of them contained functional markers of Vp1Ba and/or Dorm-B1b for resistance, the germplasm resources of Xinong 172, Kalango, Huaimai 40 and Yunong 186 contained both of functional markers. Dwarf failing rotation selection had a high efficiency in improving resistance, with 36.72% (47) of the advanced lines achieved above intermediate resistance, of which 87.23% contained resistance loci and 8 (17.02%) contained two functional loci. The accumulation of resistance loci can enhance the level of resistance. In natural populations, the whole-split germination and seed germination decreased from 36.45% and 34.99%, respectively, in non-resistant individuals to 18.17 % and 25.87 % 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, with a rate of 17.02 % for those with two markers compared to only 4.94 % for others indicating that using functional markers can significantly improve resistance. In this study, molecular markers and phenotypic characterization were combined for spike germination resistance germplasm screening, and germplasm innovation was carried out by using dwarf failing rotation selection, which provided a basis for accelerating the improvement of spike germination resistance level of wheat in the Yellow and Huaihai wheat areas.