The availability of T-DNA insertion sites is very important for plant functional genomics research and the screening and identification of transgenic plants. However, the present protocols for identifying T-DNA insertion sites, like reverse PCR and semi-random primer PCR, are not only complex and time-consuming, but also inefficient. In this paper, a DNA pool of three transgenic plants was sequenced by whole-genome resequencing, and four T-DNA insertion sites were identified by blasting using transgenic T-DNA sequences. After PCR and Southern blot analysis, the T-DNA insertion sites of the three transgenic plants were successfully confirmed, and one of the transgenic plants showed two insertion sites. In conclusion, this study established a simple, reliable and efficient method for obtaining T-DNA insertion sites in transgenic plants.