Dasypyrum villosum (2n = 14, VV) is an important tertiary gene pool of bread wheat. Excellent genes including powdery mildew resistance gene Pm55, stripe rust resistance gene Yr5v, and grain hardness gene Dina/Dinb were located on chromosome arm 5VS of D. villosum. The newly developed Triticum aestivum - Dasypyrum villosum T5VS.5AL and T5VS.5DL translocation lines provided germplasms for bread wheat improvement in disease resistance and end-use quality. In order to make efficient use of the translocation lines in wheat breeding programmes and analyze the genetic effects of the 5VS chromosome arm in wheat background, sequence information of T5VS.5DL translocated chromosome was isolated by the combination of chromosome sorting and next-generation sequencing. A total of 69 COS markers were developed by comparing the 5VS sequence information with genes on wheat ‘Chinese Spring’ homologous chromosome arms 5AS, 5BS and 5DS. After PCR amplification, 59 COS markers were identified with D. villosum 5VS specific bands, and the polymorphism rate was 85.5%, of which 40 markers were co-dominant between 5VS and homoeologous chromosome arms 5DS/5AS of wheat, and 19 markers were dominant. These markers provided information for physical mapping of beneficial genes on 5VS, and would contribute to development of small segment of 5VS in wheat. The T5VS.5AL and T5VS.5DL translocated chromosomes were introgressed into the genetic background of ‘NAU0686’ through backcross for 6 times. Comparing of powdery mildew reaction and molecular marker identification in BC6F2 generation, the reliabilities between co-dominant markers with simple amplified bands and 5VS chromosome arm were verified. In BC6F2:3 near-isoline population, the genetic effects of 5VS chromosome arm in ‘NAU0686’ background were further clarified. The results indicated that T5VS.5AL and T5VS.5DL translocated chromosomes could reduce grain hardness, plant height and 1000-grain weight, and increase grain number per main spike. The newly developed 5VS-specific molecular markers and the new germplasms laid a foundation for the efficient use of the two-translocation lines to breed new weak gluten wheat varieties with high quality and disease resistance.