Haynaldia villosa is an important genetic resource for wheat genetic improvement. The 2V chromosome in H. villosa ， which hosts many important genes， such as powdery mildew resistance， glume ridge bristles， photoperiod response， longer spikes and more grains， are valuable in common wheat improvement. However， the lack of molecular markers to the 2V chromatin impairs the introgression into wheat. In order to develop specific molecular markers on chromosome 2V， we designed two sets of primers， including： （1） 30 pairs of sequence-tagged site primers based on the expressed sequence tag sequences of different segments of the 2nd chromosome of common wheat， and （2） 296 pairs of intron targeting primers designed based on the homologous comparison between wheat 2D and rye 2R. Two and 33 specific molecular markers on chromosome 2V were validated and successfully developed， accounted for 6.7% and 11.1% of the total primers tested， respectively. This result suggests that marker development based on next generation sequencing technology is an efficient method. Most of the genes on the 2D chromosome of wheat were collinear to those of the 2V chromosome， while few exceptions were also observed， indicating a complex collinearity on the 2V chromosome of H. villosa to the 2D chromosome of common wheat. A total of 35 markers were finally qualified， including lfz8187_-1100 that was located at 2VS FL0.68-1.00， and lfz8387_-280， lfz8462_-760 and lfz8470_-200 that were located at 2VS FL0.00-0.26， as well as 31 markers that were located on the long arm of 2V chromosome. Collectively， these markers provided an effective tool for identifying structural variation of H. villosa 2V chromosome， as well as the beneficial genes introgressed into common wheat.